The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Ron Orlando - One of the best experts on this subject based on the ideXlab platform.
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On-target Exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains.
Analytical chemistry, 1999Co-Authors: Jennifer Colangelo, Ron OrlandoAbstract:One method used to determine the primary sequence of oligosaccharides is to digest them with Exoglycosidases and analyze the resulting digestion products by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous research has demonstrated that these digestions can be performed on the MALDI target. However, the procedure requires the sample to be incubated at elevated temperatures, and complete digestion requires a few hours. We demonstrate new conditions that permit Exoglycosidase digestions to be performed on the MALDI target at room temperature within 30 min. Oligosaccharide standards were digested with one or more Exoglycosidases to show that the enzymes retain their activity and specificity under these new reaction conditions. Using this method, the primary sequences of carbohydrate chains can be determined in a relatively short amount of time.
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on target Exoglycosidase digestions maldi ms for determining the primary structures of carbohydrate chains
Analytical Chemistry, 1999Co-Authors: Jennifer Colangelo, Ron OrlandoAbstract:One method used to determine the primary sequence of oligosaccharides is to digest them with Exoglycosidases and analyze the resulting digestion products by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous research has demonstrated that these digestions can be performed on the MALDI target. However, the procedure requires the sample to be incubated at elevated temperatures, and complete digestion requires a few hours. We demonstrate new conditions that permit Exoglycosidase digestions to be performed on the MALDI target at room temperature within 30 min. Oligosaccharide standards were digested with one or more Exoglycosidases to show that the enzymes retain their activity and specificity under these new reaction conditions. Using this method, the primary sequences of carbohydrate chains can be determined in a relatively short amount of time.
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Simplifying the Exoglycosidase digestion/MALDI-MS procedures for sequencing N-linked carbohydrate side chains
Analytical chemistry, 1996Co-Authors: Yi Yang, Ron OrlandoAbstract:Exoglycosidase digestion coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an effective technique for sequencing the N-linked carbohydrate side chains of a glycoprotein. However, the buffers currently used in the enzymatic procedures are detrimental to MALDI-MS, and thus desalting is required before the digestion products can be analyzed. We demonstrate that a 25 mM ammonium acetate solution adjusted to the proper pH can replace the normal Exoglycosidase digestion buffers. The use of these ammonium acetate solutions permits direct MALDI-MS analysis of the digestion mixture without desalting. More importantly, we show that many of the commonly used Exoglycosidases retain both their activity and their specificity under these conditions.
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simplifying the Exoglycosidase digestion maldi ms procedures for sequencing n linked carbohydrate side chains
Analytical Chemistry, 1996Co-Authors: Yi Yang, Ron OrlandoAbstract:Exoglycosidase digestion coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an effective technique for sequencing the N-linked carbohydrate side chains of a glycoprotein. However, the buffers currently used in the enzymatic procedures are detrimental to MALDI-MS, and thus desalting is required before the digestion products can be analyzed. We demonstrate that a 25 mM ammonium acetate solution adjusted to the proper pH can replace the normal Exoglycosidase digestion buffers. The use of these ammonium acetate solutions permits direct MALDI-MS analysis of the digestion mixture without desalting. More importantly, we show that many of the commonly used Exoglycosidases retain both their activity and their specificity under these conditions.
David J. Harvey - One of the best experts on this subject based on the ideXlab platform.
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Rapid Approach for Sequencing Neutral Oligosaccharides by Exoglycosidase Digestion and MGrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
2016Co-Authors: Bernhard Kiister, Thomas J. P. Naven, David J. HarveyAbstract:A new way of combining Exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential Exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved: (i) recording a molecular mass profile of the starting material by MALDI/TOF-MS using a mixture of 2, s dihydroxybenzoic acid and 1-hydroxyisoquinoline as the matrix; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut-off 500 Da); (iii) Exoglycosidase digestion in a volume of 1 PI; (iv) removal of the incubation buffer by droplet dialysis; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques: (i) analysis of digestion products by MALDI/TOF-MS is much faster than by chromatographic techniques; (ii) no derivatization of the analyte is required; (iii) Exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the K, of the enzyme; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G. KEY WORDS: oligosaccharides; Exoglycosidases; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; ovalbumin; immunoglobulin
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rapid approach for sequencing neutral oligosaccharides by Exoglycosidase digestion and matrix assisted laser desorption ionization time of flight mass spectrometry
Journal of Mass Spectrometry, 1996Co-Authors: Bernhard Kuster, Thomas J. P. Naven, David J. HarveyAbstract:A new way of combining Exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential Exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved : (i) recording a molecular mass profile of the starting material by MALDI/TOF-MS using a mixture of 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline as the matrix ; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut-off 500 Da) ; (iii) Exoglycosidase digestion in a volume of 1 μl ; (iv) removal of the incubation buffer by droplet dialysis ; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques : (i) analysis of digestion products by MALDI/TOF-MS is much faster than by chromatographic techniques ; (ii) no derivatization of the analyte is required ; (iii) Exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the K m of the enzyme ; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G.
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Rapid Approach for Sequencing Neutral Oligosaccharides by Exoglycosidase Digestion and Matrix‐assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry
Journal of mass spectrometry : JMS, 1996Co-Authors: Bernhard Kuster, Thomas J. P. Naven, David J. HarveyAbstract:A new way of combining Exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential Exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved : (i) recording a molecular mass profile of the starting material by MALDI/TOF-MS using a mixture of 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline as the matrix ; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut-off 500 Da) ; (iii) Exoglycosidase digestion in a volume of 1 μl ; (iv) removal of the incubation buffer by droplet dialysis ; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques : (i) analysis of digestion products by MALDI/TOF-MS is much faster than by chromatographic techniques ; (ii) no derivatization of the analyte is required ; (iii) Exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the K m of the enzyme ; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G.
Bernhard Kuster - One of the best experts on this subject based on the ideXlab platform.
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rapid approach for sequencing neutral oligosaccharides by Exoglycosidase digestion and matrix assisted laser desorption ionization time of flight mass spectrometry
Journal of Mass Spectrometry, 1996Co-Authors: Bernhard Kuster, Thomas J. P. Naven, David J. HarveyAbstract:A new way of combining Exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential Exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved : (i) recording a molecular mass profile of the starting material by MALDI/TOF-MS using a mixture of 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline as the matrix ; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut-off 500 Da) ; (iii) Exoglycosidase digestion in a volume of 1 μl ; (iv) removal of the incubation buffer by droplet dialysis ; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques : (i) analysis of digestion products by MALDI/TOF-MS is much faster than by chromatographic techniques ; (ii) no derivatization of the analyte is required ; (iii) Exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the K m of the enzyme ; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G.
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Rapid Approach for Sequencing Neutral Oligosaccharides by Exoglycosidase Digestion and Matrix‐assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry
Journal of mass spectrometry : JMS, 1996Co-Authors: Bernhard Kuster, Thomas J. P. Naven, David J. HarveyAbstract:A new way of combining Exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential Exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved : (i) recording a molecular mass profile of the starting material by MALDI/TOF-MS using a mixture of 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline as the matrix ; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut-off 500 Da) ; (iii) Exoglycosidase digestion in a volume of 1 μl ; (iv) removal of the incubation buffer by droplet dialysis ; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques : (i) analysis of digestion products by MALDI/TOF-MS is much faster than by chromatographic techniques ; (ii) no derivatization of the analyte is required ; (iii) Exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the K m of the enzyme ; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G.
Takao Hayakawa - One of the best experts on this subject based on the ideXlab platform.
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Plug-plug kinetic capillary electrophoresis for in-capillary Exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans.
Journal of chromatography. A, 2017Co-Authors: Maki Yamagami, Sachio Yamamoto, Takao Hayakawa, Yurie Matsui, Mitsuhiro Kinoshita, Shigeo SuzukiAbstract:An online Exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An Exoglycosidase solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer solution containing 0.5w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of APTS-labeled oligosaccharides are released by the action of an Exoglycosidase, the migration times of the oligosaccharides shift to those of digested oligosaccharides. We examined β-galactosidase, α-mannosidase, β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, and found only β-galactosidase and α-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the reaction was completed by injecting a 3.6cm long plug of 200 and 50mU/mL concentration of Exoglycosidases. In contrast, other Exoglycosidases could not react with APTS labeled oligosaccharides at a concentration up to 5U/mL. The β-N-acetylhexosaminidase reaction was successively followed by the electrophoretic mobility of APTS oligosaccharides and stopped for 10min when saccharide derivatives were achieved in the enzyme plug. The reaction of α-fucosidase and α-mannosidase was completed by decreasing the electrophoretic voltage to -2kV when the APTS oligosaccharides were passing through an Exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of glycoprotein glycans.
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Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody.
Electrophoresis, 2011Co-Authors: Yuki Yagi, Sachio Yamamoto, Yukihito Ohyama, Takao Hayakawa, Shigeo SuzukiAbstract:: Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and Exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab. In the lectin-filling method, Galb1–4GlcNAc-specific Erythrina cristagali agglutinin, a1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Aca2–3Gal-specific Maackia amurensis lectin were used. The oligosaccharides migrated through the lectin plug during separation; the changes in separation profiles were observed according to the interaction with the lectins. The glycosidase-filling method allowed rapid digestion as suggested by the electropherograms. Partial-filling CE methods can avoid tedious hands-on procedures such as overnight incubation and optimization reaction condition with lectins and Exoglycosidases. Combination of these partial-filling capillary electrophoresis methods makes the characterization of oligosaccharide profiles of therapeutic antibodies easier and faster.
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Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidas
Analytical biochemistry, 2009Co-Authors: Mitsuhiro Kinoshita, Hiroko Ohta, Kanata Higaki, Yoko Kojima, Tadasu Urashima, Kazuki Nakajima, Minoru Suzuki, Kit M. Kovacs, Christian Lydersen, Takao HayakawaAbstract:Abstract A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW ∼ 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase–high-performance liquid chromatography–matrix-assisted laser desorption/ionization–time-of-flight (NP–HPLC–MALDI–TOF) mass spectrometry (MS) by combination with sequential Exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI–TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galβ1–4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with Exoglycosidases and characterized by MALDI–TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto- N -neohexaose (LNnH, Galβ1–4GlcNAcβ1–6[Galβ1–4GlcNAcβ1–3]Galβ1–4Glc) as the common core structure, and most of them contained Fucα1–2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1–4GlcNAc ( N -acetyllactosamine, LacNAc) residues on the Galβ1–4GlcNAcβ1–3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N -acetyllactosamine at the 6-OH position.
Rudolf Geyer - One of the best experts on this subject based on the ideXlab platform.
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The Liver Flukes Fasciola gigantica and Fasciola hepatica Express the Leucocyte Cluster of Differentiation Marker CD77 (Globotriaosylceramide) in Their Tegument
Biological chemistry, 2001Co-Authors: Manfred Wuhrer, Clemens M. Berkefeld, Roger D. Dennis, Mohamed A. Idris, Rudolf GeyerAbstract:Glycosphingolipids from the parasitic liver flukes Fasciola gigantica and Fasciola hepatica were isolated and their carbohydrate moieties were structurally analysed by methylation analysis, Exoglycosidase treatment, on-target Exoglycosidase cleavage and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. For both liver fluke species, the ceramide monohexosides Gal1-ceramide and Glc1-ceramide were found in relative amounts of 1.0 to 0.1, respectively. From F. gigantica, the ceramide dihexoside was isolated in sufficient amounts to be structurally determined as lactosylceramide, Gal beta4-Glc1-ceramide, while for both liver fluke species the ceramide trihexoside was shown to be Gal alpha4Gal beta4-Glc1-ceramide, which is designated as either globotriaosylceramide, Pk-blood group antigen or CD77 leucocyte cluster of differentiation antigen. To our knowledge, this is the first report on the expression of globo-series glycosphingolipids in non-mammalian species. Ceramide analysis of ceramide monohexosides yielded as major components octadecanoic and 2-hydroxyoctadecanoic fatty acids together with C18- and C20-phytosphingosines. By the use of an anti-CD77 monoclonal antibody and the Escherichia coli Shiga toxin B1 subunit, globotriaosylceramide could be immunolocalised to the tegument of F. hepatica cryosections. The sharing of CD77 between liver flukes and their mammalian hosts fits in with the concept of molecular mimicry, which is closely parallel to the established imitation of host CD15 (Lewis X) displayed by the blood fluke Schistosoma mansoni.
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Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS.
Analytical chemistry, 1999Co-Authors: Hildegard Geyer, Manfred Wuhrer, Sigrid Schmitt, Rudolf GeyerAbstract:Exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been demonstrated to be an effective method for the structural characterization of glycoconjugates and oligosaccharides in picomolar amounts. A sample preparation method is described, in which 6-aza-2-thiothymine (ATT) in water is used as matrix and enzymes are dialyzed before use against a low concentration of volatile buffer such as ammonium acetate. Under these conditions, a series of sequential on-target Exoglycosidase treatments was carried out in one single analyte spot in the presence of ATT matrix. Subsequent mass spectrometric analysis of the resulting products yielded information on both the completeness of the reaction and structural features of the glycoconjugates such as monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. The results show that all Exoglycosidases used retain their activity in the pr...