The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Niclas G. Karlsson - One of the best experts on this subject based on the ideXlab platform.
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Mass spectrometric analysis of O-linked Oligosaccharides from various recombinant expression systems.
Methods of Molecular Biology, 2013Co-Authors: Diarmuid T. Kenny, Stefan Gaunitz, Catherine A. Hayes, Anki Gustafsson, Magnus Sjöblom, Jan Holgersson, Niclas G. KarlssonAbstract:Analysis of O-linked glycosylation is one of the main challenges during structural validation of recombinant glycoproteins. With methods available for N-linked glycosylation in regard to Oligosaccharide analysis as well as glycopeptide mapping, there are still challenges for O-linked glycan analysis. Here, we present mass spectrometric methodology for O-linked Oligosaccharides released by reductive β-elimination. Using LC-MS and LC-MS(2) with graphitized carbon columns, Oligosaccharides are analyzed without derivatization. This approach provides a high-throughput method for screening during clonal selection, as well as product structure verification, without impairing sequencing ability. The protocols are exemplified by analysis of glycoproteins from mammalian cell cultures (CHO cells) as well as insect cells and yeast. The data shows that the method can be successfully applied to both neutral and acidic O-linked Oligosaccharides, where sialic acid, hexuronic acid, and sulfate are common substituents. Further characterization of O-glycans can be achieved using permethylation. Permethylation of O-linked Oligosaccharides followed by direct infusion into the mass spectrometer provide information about Oligosaccharide composition, and subsequent MS (n) experiments can be carried out to elucidate Oligosaccharide structure including linkage information and sequence.
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Small-scale enzymatic digestion of glycoproteins and proteoglycans for analysis of Oligosaccharides by LC-MS and FACE gel electrophoresis.
Methods in molecular biology (Clifton N.J.), 2008Co-Authors: Ruby P. Estrella, John M. Whitelock, Rebecca H. Roubin, Nicolle H. Packer, Niclas G. KarlssonAbstract:Structural characterization of Oligosaccharides from proteoglycans and other glycoproteins is greatly enhanced through the use of mass spectrometry and gel electrophoresis. Sample preparation for these sensitive techniques often requires enzymatic treatments to produce Oligosaccharide sequences for subsequent analysis. This chapter describes several small-scale methods for in-gel, on-blot, and in-solution enzymatic digestions in preparation for graphitized carbon liquid chromatography-mass spectrometry (LC-MS) analysis, with specific applications indicated for glycosaminoglycans (GAGs) and N-linked Oligosaccharides. In addition, accompanying procedures for Oligosaccharide reduction by sodium borohydride, sample desalting via carbon microcolumn, desialylation by sialidase enzyme treatment, and small-scale Oligosaccharide species fractionation are included. Fluorophore-assisted carbohydrate electrophoresis (FACE) is another useful method to isolate derivatized Oligosaccharides. Overall, the modularity of these techniques provides ease and flexibility for use in conjunction with mass spectrometric and electrophoretic tools for glycomic research studies.
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development of a mass fingerprinting tool for automated interpretation of Oligosaccharide fragmentation data
Proteomics, 2004Co-Authors: Hiren J Joshi, Nicolle H. Packer, Mathew J Harrison, B Schulz, Catherine A Cooper, Niclas G. KarlssonAbstract:The bioinformatic tool GlycosidIQTM was developed for computerized interpretation of Oligosaccharide mass spectrometric fragmentation based on matching experimental data with theoretically fragmented Oligosaccharides generated from the database GlycoSuiteDBTM. This use of the software for glycofragment mass fingerprinting obviates a large part of the manual, labor intensive, and technically challenging interpretation of Oligosaccharide fragmentation. Using 130 negative ion electrospray ionization-tandem mass spectrometry fragment spectra from identified Oligosaccharide structures, it was shown that the GlycosidIQ scoring algorithms were able to correctly identify Oligosaccharides in the great majority of cases (correct structure top ranked in 78% of the cases and an additional 17% were ranked second highest in the sample set).
Shaohong Jin - One of the best experts on this subject based on the ideXlab platform.
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Structural characterization of N-linked Oligosaccharides of Defibrase from Agikistrodon acutus by sequential exoglycosidase digestion and MALDI-TOF mass spectrometry
Toxicon, 2009Co-Authors: Xiao-qing Luo, Huaxin Yang, Chenggang Liang, Shaohong JinAbstract:Detailed structures of N-linked Oligosaccharides of Defibrase, a highly active thrombin like enzyme (TLE) purified from the venom of Agkistrodon acutus, were successfully characterized using MALDI-TOF mass spectrometry in combination with sequential exoglycosidase digestion. Monosaccharide composition analysis was performed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Galactose(Gal), mannose(Man), fucose(Fuc), N-acetylglucosamine (GlcNAc), and sialic acid (Neu5Ac) was detected and the total carbohydrate content was about 19.4% (w/w). The N-linked Oligosaccharides were released by treatment with PNGase F, fluorescent labeled with 2-aminobenzamide, and fractionated by high performance liquid chromatography (HPLC). The main Oligosaccharide fractions were collected and further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the Oligosaccharide. The combined data revealed the presence of five distinct Oligosaccharide structures in Defibrase, which are mainly complex or hybrid type, with a small amount of oligomannosidic type. The complex type Oligosaccharides are mostly tri-or bi-antennary and the hybrid Oligosaccharides are all bi-antennary. Most Oligosaccharides are also found to be fucosylated.
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Structural characterization of N-linked Oligosaccharides of Defibrase from Agkistrodon acutus by sequential exoglycosidase digestion and MALDI-TOF mass spectrometry.
Toxicon : official journal of the International Society on Toxinology, 2009Co-Authors: Xiao-qing Luo, Huaxin Yang, Chenggang Liang, Shaohong JinAbstract:Detailed structures of N-linked Oligosaccharides of Defibrase, a highly active thrombin like enzyme (TLE) purified from the venom of Agkistrodon acutus, were successfully characterized using MALDI-TOF mass spectrometry in combination with sequential exoglycosidase digestion. Monosaccharide composition analysis was performed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Galactose(Gal), mannose(Man), fucose(Fuc), N-acetylglucosamine (GlcNAc), and sialic acid (Neu5Ac) was detected and the total carbohydrate content was about 19.4% (w/w). The N-linked Oligosaccharides were released by treatment with PNGase F, fluorescent labeled with 2-aminobenzamide, and fractionated by high performance liquid chromatography (HPLC). The main Oligosaccharide fractions were collected and further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the Oligosaccharide. The combined data revealed the presence of five distinct Oligosaccharide structures in Defibrase, which are mainly complex or hybrid type, with a small amount of oligomannosidic type. The complex type Oligosaccharides are mostly tri-or bi-antennary and the hybrid Oligosaccharides are all bi-antennary. Most Oligosaccharides are also found to be fucosylated.
Tsuguo Mizuochi - One of the best experts on this subject based on the ideXlab platform.
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β subunits of equine chorionic gonadotropin and lutenizing hormone with an identical amino acid sequence have different asparagine linked Oligosaccharide chains
Biochemical and Biophysical Research Communications, 1991Co-Authors: Taei Matsui, George R. Bousfield, Darrell N Ward, Hiromu Sugino, Mari Miura, Koiti Titani, Tsuguo MizuochiAbstract:Abstract The glycoprotein hormones, equine chorionic gonadotropin (eCG) and lutenizing hormone (eLH), possess a β-subunit with an identical amino acid sequence. The Asn-linked Oligosaccharide chains of eCGβ and eLHβ were quantitatively liberated as tritium-labeled Oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4-reduction. Paper electrophoresis in combination with sialidase digestion and solvolytic desulfation indicated that eCGβ contained neutral and sialylated Oligosaccharides, while eLHβ contained neutral, sialylated, sulfated, and both sialylated and sulfated Oligosaccharides. In addition, elution profiles on a Bio-Gel P-4 column of the neutralized Oligosaccharide mixtures of eCGβ and eLHβ were different, indicating that the molecular masses of Oligosaccharides of the two glycoproteins are different. Therefore, this suggests that the structures of the Asn-linked Oligosaccharide chains of eCGβ and eLHβ are different although they have an identical amino acid sequence.
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β subunits of equine chorionic gonadotropin and lutenizing hormone with an identical amino acid sequence have different asparagine linked Oligosaccharide chains
Biochemical and Biophysical Research Communications, 1991Co-Authors: Taei Matsui, George R. Bousfield, Darrell N Ward, Hiromu Sugino, Mari Miura, Koiti Titani, Tsuguo MizuochiAbstract:The glycoprotein hormones, equine chorionic gonadotropin (eCG) and lutenizing hormone (eLH), possess a beta-subunit with an identical amino acid sequence. The Asn-linked Oligosaccharide chains of eCG beta and eLH beta were quantitatively liberated as tritium-labeled Oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4-reduction. Paper electrophoresis in combination with sialidase digestion and solvolytic desulfation indicated that eCG beta contained neutral and sialylated Oligosaccharides, while eLH beta contained neutral, sialylated, sulfated, and both sialylated and sulfated Oligosaccharides. In addition, elution profiles on a Bio-Gel P-4 column of the neutralized Oligosaccharide mixtures of eCG beta and eLH beta were different, indicating that the molecular masses of Oligosaccharides of the two glycoproteins are different. Therefore, this suggests that the structures of the Asn-linked Oligosaccharide chains of eCG beta and eLH beta are different although they have an identical amino acid sequence.
Andreas H. Franz - One of the best experts on this subject based on the ideXlab platform.
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MALDI-TOF and ESI-MS analysis of Oligosaccharides labeled with a new multifunctional Oligosaccharide tag
Journal of The American Society for Mass Spectrometry, 2006Co-Authors: Joanne Hsu, Soo Jin Chang, Andreas H. FranzAbstract:A new multifunctional Oligosaccharide label with a 1^o amino-group was synthesized and characterized. The Oligosaccharide label was introduced into several neutral Oligosaccharides by reductive amination, and the derivatives were analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and by electrospray ionization (ESI) mass spectrometry. It was demonstrated that the labeling reaction was satisfactory, and that as little as 50 pmol of starting material could be efficiently labeled with minimal loss to side reactions. A mixture of high-mannose N -glycans released from ribonuclease B was labeled. The label did not appear to interfere with structural characterization of the Oligosaccharides by mass spectrometry. N -quaternization of the labeled Oligosaccharides resulted in significantly increased sensitivity of detection with as little as 100 fmol on the probe detected. Deuterium coding of labeled Oligosaccharide mixtures and relative abundance of mixture components was investigated. A protocol for the chromatographic separation of mixtures of labeled Oligosaccharides by HPLC was developed and is reported here.
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MALDI-TOF and ESI-MS analysis of Oligosaccharides labeled with a new multifunctional Oligosaccharide tag.
Journal of the American Society for Mass Spectrometry, 2006Co-Authors: Joanne Hsu, Soo Jin Chang, Andreas H. FranzAbstract:A new multifunctional Oligosaccharide label with a 1 degree amino-group was synthesized and characterized. The Oligosaccharide label was introduced into several neutral Oligosaccharides by reductive amination, and the derivatives were analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and by electrospray ionization (ESI) mass spectrometry. It was demonstrated that the labeling reaction was satisfactory, and that as little as 50 pmol of starting material could be efficiently labeled with minimal loss to side reactions. A mixture of high-mannose N-glycans released from ribonuclease B was labeled. The label did not appear to interfere with structural characterization of the Oligosaccharides by mass spectrometry. N-quaternization of the labeled Oligosaccharides resulted in significantly increased sensitivity of detection with as little as 100 fmol on the probe detected. Deuterium coding of labeled Oligosaccharide mixtures and relative abundance of mixture components was investigated. A protocol for the chromatographic separation of mixtures of labeled Oligosaccharides by HPLC was developed and is reported here.
Y Jigami - One of the best experts on this subject based on the ideXlab platform.
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structure of the n linked Oligosaccharides that show the complete loss of alpha 1 6 polymannose outer chain from och1 och1 mnn1 and och1 mnn1 alg3 mutants of saccharomyces cerevisiae
Journal of Biological Chemistry, 1993Co-Authors: Y Nakanishishindo, Kenichi Nakayama, Atsushi Tanaka, Y Toda, Y JigamiAbstract:Abstract The periplasmic invertase was purified from Saccharomyces cerevisiae och1::LEU2 disruptant cells (delta och1), which have a defect in elongation of the outer chain attached to the N-linked core Oligosaccharides (Nakayama, K., Nagasu, T., Shimma, Y., Kuromatsu, J., and Jigami, Y. (1992) EMBO J. 11, 2511-2519). Structural analysis of the pyridylaminated (PA) neutral Oligosaccharides released by hydrazinolysis and N-acetylation confirmed that the och1 mutation causes a complete loss of the alpha-1,6-polymannose outer chain, although the PA Oligosaccharides (Man9GlcNAc2-PA and Man10GlcNAc2-PA), in which one or two alpha-1,3-linked mannose(s) attached to the endoplasmic reticulumn (ER)-form core Oligosaccharide (Man8GlcNAc2) were also detected. Analysis of the delta och1 mnn1 strain Oligosaccharides released from total cell mannoprotein revealed that the delta och1 mnn1 mutant eliminates the alpha-1,3-mannose attached to the core and accumulates predominantly a single ER-form Oligosaccharide species (Man8GlcNAc2), suggesting a potential use of this strain as a host cell to produce glycoproteins containing mammalian high mannose type Oligosaccharides. The delta och1 mnn1 alg3 mutants accumulated Man5GlcNAc2 and Man8GlcNAc2 in total cell mannoprotein, confirming the lack of outer chain addition to the incomplete corelike Oligosaccharide and the leaky phenotype of the alg3 mutation. All the results suggest that the OCH1 gene encodes an alpha-1,6-mannosyltransferase that is functional in the initiation of alpha-1,6-polymannose outer chain addition to the N-linked core Oligosaccharide (Man5GlcNAc2 and Man8GlcNAc2) in yeast.
