The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Patrick Camilleri - One of the best experts on this subject based on the ideXlab platform.
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Early indication of effects of puromycin aminonucleoside using a fluorimetric assay of 2-Aminoacridone-derivatized carbohydrates in urine.
Analytical biochemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, David Tolson, John Connelly, Patrick CamilleriAbstract:This paper describes a novel noninvasive method to study the changes in free carbohydrates excreted in urine as a result of toxicity in the rat induced by the administration of puromycin aminonucleoside (PAN). Urine samples were collected for 24 h prior to dosing and at 8, 24, and 32 h postdosing. For each sample, free carbohydrates were extracted from the urine using a graphitized carbon column and then labeled with 2-Aminoacridone (2-AMAC) prior to analysis by hydrophilic interaction liquid chromatography (HILC). Dramatic changes were seen in the profile of the carbohydrates at the 8- and 24-h time points. These changes in carbohydrate profiles may be useful as early indicators of toxicity.
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Analysis of oligosaccharides by microbore high-performance liquid chromatography.
Analytical chemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, Jim Langridge, Edouard S. P. Bouvier, Patrick CamilleriAbstract:A 1-mm microbore hydrophilic interaction column has been used for the separation of 2-Aminoacridone (2-AMAC)-derivatized glycan mixtures, released from naturally occurring and recombinant proteins. Primary structure identification of the 2-AMAC glycan derivatives was carried out by HPLC using fluorescence and mass spectrometric detection. In some cases, enzymatic digestion of the 2-AMAC glycans was applied to confirm glycan structure. This strategy is considerably more rapid than methods normally used in glycan analysis, which involves manual collection of separated oligosaccharide derivatives and analysis of individual fractions by mass spectrometry.
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A probe for the versatile analysis and characterization of N-linked oligosaccharides.
Analytical chemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, David Tolson, Andy Gribble, Vincent Burdes, Patrick CamilleriAbstract:A novel fluorescent probe, 3-(acetylamino)-6-aminoacridine (AA-Ac), has been synthesized and its applicability to the analysis of picomole levels of N-linked glycans investigated. AA-Ac was found to be an excellent derivatization reagent for N-linked glycans, giving at least twice the intensity of fluorescence as its predecessor 2-Aminoacridone. AA-Ac-labeled glycans were analyzed by both normal and reversed-phase HPLC. They were also amenable to enzymatic sequencing and analysis by MALDI-TOF mass spectrometry, free zone capillary electrophoresis, and capillary electrophoresis/electrospray ionization mass spectrometry.
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Carbohydrate release from picomole quantities of glycoprotein and characterisation of glycans by high-performance liquid chromatography and mass spectrometry
Journal of chromatography. B Biomedical sciences and applications, 1999Co-Authors: Joanne Charlwood, Helen Birrell, Patrick CamilleriAbstract:Samples of 5 to 20 μg of human IgG were subjected to dithiothreitol treatment to reduce disulphide bridges, followed by tryptic digestion. Glycans released from the tryptic peptide mixture by PNGase F digestion were then derivatised with 2-Aminoacridone. Labelled oligosaccharides were separated by normal-phase high-performance liquid chromatography and individual components were collected for matrix-assisted laser desorption ionization time-of-flight and electrospray mass spectrometric analysis.
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Structural characterisation of N-linked glycan mixtures by precursor ion scanning and tandem mass spectrometric analysis
Rapid communications in mass spectrometry : RCM, 1999Co-Authors: Joanne Charlwood, Jim Langridge, Patrick CamilleriAbstract:2-Aminoacridone (2-AMAC) labelled N-linked glycan pools were analysed directly by a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-Tof) in the precursor ion scanning and tandem mass spectrometry (MS/MS) modes. The use of a precursor ion scanning strategy on this instrument provides a rapid and sensitive method of screening glycan mixtures, without prior separation by chromatographic methods. It allows facile and preliminary characterisation of glycans into different classes, for example, high-mannose or complex glycans. Preliminary sequencing information for each glycan is obtained in the initial precursor ion scanning mode, but further sequencing information of selected glycans can be obtained using the MS/MS mode. Copyright 1999 John Wiley & Sons, Ltd.
Nicola Volpi - One of the best experts on this subject based on the ideXlab platform.
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Analysis of glycosaminoglycan-derived, precolumn, 2-Aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection
Nature Protocols, 2014Co-Authors: Nicola Volpi, Fabio Galeotti, Bo Yang, Robert J. LinhardtAbstract:Glycosaminoglycans (GAGs) possess considerable heterogeneity in average molecular mass, molecular mass range, disaccharide composition and content and position of sulfo groups. Despite recent technological advances in the analysis of GAGs, the determination of GAG disaccharide composition still remains challenging and provides key information required for understanding GAG function. Analysis of GAG-derived disaccharides relies on enzymatic treatment, providing one of the most practical and quantitative approaches for compositional mapping. Tagging the reducing end of disaccharides with an aromatic fluorescent label affords stable derivatives with properties that enable improved detection and resolution. HPLC with on-line electrospray ionization mass spectrometry (ESI-MS) offers a relatively soft ionization method for detection and characterization of sulfated oligosaccharides. GAGs obtained from tissues, biological fluids or cells are treated with various enzymes to obtain disaccharides that are fluorescently labeled with 2-Aminoacridone (AMAC) and resolved by different LC systems for high-sensitivity detection by fluorescence, and then they are unambiguously characterized by MS. The preparation and labeling of GAG-derived disaccharides can be performed in ∼1–2 d, and subsequent HPLC separation and on-line fluorescence detection and ESI-MS analysis takes another 1–2 h.
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analysis of glycosaminoglycan derived precolumn 2 aminoacridone labeled disaccharides with lc fluorescence and lc ms detection
Nature Protocols, 2014Co-Authors: Nicola Volpi, Fabio Galeotti, Bo Yang, Robert J. LinhardtAbstract:Analysis of glycosaminoglycan-derived, precolumn, 2-Aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection
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Analysis of glycosaminoglycan-derived, precolumn, 2-Aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection
Nature protocols, 2014Co-Authors: Nicola Volpi, Fabio Galeotti, Bo Yang, Robert J. LinhardtAbstract:Analysis of glycosaminoglycan-derived, precolumn, 2-Aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection
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Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2‐aminoacridone
Electrophoresis, 2014Co-Authors: Fabio Galeotti, Francesca Maccari, Giovanni V. Coppa, Lucia Zampini, Tiziana Galeazzi, Lucia Padella, Lucia Santoro, Orazio Gabrielli, Nicola VolpiAbstract:Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high-voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2-Aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on-capillary anionic borate-polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ∼50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.
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online reverse phase high performance liquid chromatography fluorescence detection electrospray ionization mass spectrometry separation and characterization of heparan sulfate heparin and low molecular weight heparin disaccharides derivatized with 2
Analytical Chemistry, 2011Co-Authors: Fabio Galeotti, Nicola VolpiAbstract:A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-Aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ∼15–21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of ...
Helen Birrell - One of the best experts on this subject based on the ideXlab platform.
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Early indication of effects of puromycin aminonucleoside using a fluorimetric assay of 2-Aminoacridone-derivatized carbohydrates in urine.
Analytical biochemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, David Tolson, John Connelly, Patrick CamilleriAbstract:This paper describes a novel noninvasive method to study the changes in free carbohydrates excreted in urine as a result of toxicity in the rat induced by the administration of puromycin aminonucleoside (PAN). Urine samples were collected for 24 h prior to dosing and at 8, 24, and 32 h postdosing. For each sample, free carbohydrates were extracted from the urine using a graphitized carbon column and then labeled with 2-Aminoacridone (2-AMAC) prior to analysis by hydrophilic interaction liquid chromatography (HILC). Dramatic changes were seen in the profile of the carbohydrates at the 8- and 24-h time points. These changes in carbohydrate profiles may be useful as early indicators of toxicity.
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A probe for the versatile analysis and characterization of N-linked oligosaccharides.
Analytical chemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, David Tolson, Andy Gribble, Vincent Burdes, Patrick CamilleriAbstract:A novel fluorescent probe, 3-(acetylamino)-6-aminoacridine (AA-Ac), has been synthesized and its applicability to the analysis of picomole levels of N-linked glycans investigated. AA-Ac was found to be an excellent derivatization reagent for N-linked glycans, giving at least twice the intensity of fluorescence as its predecessor 2-Aminoacridone. AA-Ac-labeled glycans were analyzed by both normal and reversed-phase HPLC. They were also amenable to enzymatic sequencing and analysis by MALDI-TOF mass spectrometry, free zone capillary electrophoresis, and capillary electrophoresis/electrospray ionization mass spectrometry.
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Analysis of oligosaccharides by microbore high-performance liquid chromatography.
Analytical chemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, Jim Langridge, Edouard S. P. Bouvier, Patrick CamilleriAbstract:A 1-mm microbore hydrophilic interaction column has been used for the separation of 2-Aminoacridone (2-AMAC)-derivatized glycan mixtures, released from naturally occurring and recombinant proteins. Primary structure identification of the 2-AMAC glycan derivatives was carried out by HPLC using fluorescence and mass spectrometric detection. In some cases, enzymatic digestion of the 2-AMAC glycans was applied to confirm glycan structure. This strategy is considerably more rapid than methods normally used in glycan analysis, which involves manual collection of separated oligosaccharide derivatives and analysis of individual fractions by mass spectrometry.
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Carbohydrate release from picomole quantities of glycoprotein and characterisation of glycans by high-performance liquid chromatography and mass spectrometry
Journal of chromatography. B Biomedical sciences and applications, 1999Co-Authors: Joanne Charlwood, Helen Birrell, Patrick CamilleriAbstract:Samples of 5 to 20 μg of human IgG were subjected to dithiothreitol treatment to reduce disulphide bridges, followed by tryptic digestion. Glycans released from the tryptic peptide mixture by PNGase F digestion were then derivatised with 2-Aminoacridone. Labelled oligosaccharides were separated by normal-phase high-performance liquid chromatography and individual components were collected for matrix-assisted laser desorption ionization time-of-flight and electrospray mass spectrometric analysis.
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A chromatographic and mass spectrometric strategy for the analysis of oligosaccharides: determination of the glycan structures in porcine thyroglobulin.
Rapid communications in mass spectrometry : RCM, 1999Co-Authors: Joanne Charlwood, Helen Birrell, Andrew J. Organ, Patrick CamilleriAbstract:Oligosaccharides released from porcine thyroglobulin were first derivatised with 2-Aminoacridone (2-AMAC) and analysed by capillary electrophoresis to determine the complexity of this glycan pool. The same glycan mixture was then subjected to either a sialidase digest or a sialidase and fucosidase digest prior to derivatisation with 2-AMAC and analysis by normal phase high pressure liquid chromatography (HPLC). Comparison of the three chromatographic profiles with known standards allowed an initial identification of the glycan structures. The 2-AMAC derivatised glycans were then collected from HPLC for matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) analysis and the molecular weights of predicted structures were confirmed. This study demonstrates that a two enzyme array and subsequent MALDI-TOF analysis can be used successfully to assign the major glycans present in a complex mixture.
Joanne Charlwood - One of the best experts on this subject based on the ideXlab platform.
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Early indication of effects of puromycin aminonucleoside using a fluorimetric assay of 2-Aminoacridone-derivatized carbohydrates in urine.
Analytical biochemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, David Tolson, John Connelly, Patrick CamilleriAbstract:This paper describes a novel noninvasive method to study the changes in free carbohydrates excreted in urine as a result of toxicity in the rat induced by the administration of puromycin aminonucleoside (PAN). Urine samples were collected for 24 h prior to dosing and at 8, 24, and 32 h postdosing. For each sample, free carbohydrates were extracted from the urine using a graphitized carbon column and then labeled with 2-Aminoacridone (2-AMAC) prior to analysis by hydrophilic interaction liquid chromatography (HILC). Dramatic changes were seen in the profile of the carbohydrates at the 8- and 24-h time points. These changes in carbohydrate profiles may be useful as early indicators of toxicity.
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A probe for the versatile analysis and characterization of N-linked oligosaccharides.
Analytical chemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, David Tolson, Andy Gribble, Vincent Burdes, Patrick CamilleriAbstract:A novel fluorescent probe, 3-(acetylamino)-6-aminoacridine (AA-Ac), has been synthesized and its applicability to the analysis of picomole levels of N-linked glycans investigated. AA-Ac was found to be an excellent derivatization reagent for N-linked glycans, giving at least twice the intensity of fluorescence as its predecessor 2-Aminoacridone. AA-Ac-labeled glycans were analyzed by both normal and reversed-phase HPLC. They were also amenable to enzymatic sequencing and analysis by MALDI-TOF mass spectrometry, free zone capillary electrophoresis, and capillary electrophoresis/electrospray ionization mass spectrometry.
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Analysis of oligosaccharides by microbore high-performance liquid chromatography.
Analytical chemistry, 2000Co-Authors: Joanne Charlwood, Helen Birrell, Jim Langridge, Edouard S. P. Bouvier, Patrick CamilleriAbstract:A 1-mm microbore hydrophilic interaction column has been used for the separation of 2-Aminoacridone (2-AMAC)-derivatized glycan mixtures, released from naturally occurring and recombinant proteins. Primary structure identification of the 2-AMAC glycan derivatives was carried out by HPLC using fluorescence and mass spectrometric detection. In some cases, enzymatic digestion of the 2-AMAC glycans was applied to confirm glycan structure. This strategy is considerably more rapid than methods normally used in glycan analysis, which involves manual collection of separated oligosaccharide derivatives and analysis of individual fractions by mass spectrometry.
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Carbohydrate release from picomole quantities of glycoprotein and characterisation of glycans by high-performance liquid chromatography and mass spectrometry
Journal of chromatography. B Biomedical sciences and applications, 1999Co-Authors: Joanne Charlwood, Helen Birrell, Patrick CamilleriAbstract:Samples of 5 to 20 μg of human IgG were subjected to dithiothreitol treatment to reduce disulphide bridges, followed by tryptic digestion. Glycans released from the tryptic peptide mixture by PNGase F digestion were then derivatised with 2-Aminoacridone. Labelled oligosaccharides were separated by normal-phase high-performance liquid chromatography and individual components were collected for matrix-assisted laser desorption ionization time-of-flight and electrospray mass spectrometric analysis.
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Structural characterisation of N-linked glycan mixtures by precursor ion scanning and tandem mass spectrometric analysis
Rapid communications in mass spectrometry : RCM, 1999Co-Authors: Joanne Charlwood, Jim Langridge, Patrick CamilleriAbstract:2-Aminoacridone (2-AMAC) labelled N-linked glycan pools were analysed directly by a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-Tof) in the precursor ion scanning and tandem mass spectrometry (MS/MS) modes. The use of a precursor ion scanning strategy on this instrument provides a rapid and sensitive method of screening glycan mixtures, without prior separation by chromatographic methods. It allows facile and preliminary characterisation of glycans into different classes, for example, high-mannose or complex glycans. Preliminary sequencing information for each glycan is obtained in the initial precursor ion scanning mode, but further sequencing information of selected glycans can be obtained using the MS/MS mode. Copyright 1999 John Wiley & Sons, Ltd.
Robert J. Linhardt - One of the best experts on this subject based on the ideXlab platform.
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Capillary Electrophoresis for the Analysis
2015Co-Authors: Of Glycosaminoglycan-derived Disaccharides, Amanda Weyers, Yuqing Chang, Bo Yang, Robert J. LinhardtAbstract:Capillary electrophoresis is a common technique used for glycosaminoglycan-derived disaccharide analysis because of its high resolving power, high separation ef fi ciency, high sensitivity, short analysis time, and straightforward operation. CE coupled to laser-induced fl uorescence (LIF) detection shows an approxi-mately 100 times higher sensitivity than traditional UV detection at 232 nm. 2-Aminoacridone (AMAC) is a widely used fl uorophore for labeling unsaturated disaccharides by deductive amination, which is one of the most important method of derivatization of disaccharides for CE-LIF detection. Outlined in this chapter is a protocol of analyzing glycosaminoglycan-derived disaccharides by CE-LIF with AMAC derivatization
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Glycosaminoglycanomics of Cultured Cells Using a Rapid and Sensitive LC-MS/MS Approach
ACS chemical biology, 2015Co-Authors: Fang Tian, Linxia Zhang, Changhu Xue, Robert J. LinhardtAbstract:Glycosaminoglycans (GAGs), a family of polysaccharides widely distributed in eukaryotic cells, are responsible for a wide array of biological functions. Quantitative disaccharide compositional analysis is one of the primary ways to characterize the GAG structure. This structural analysis is typically time-consuming (1–2 weeks) and labor intensive, requiring GAG recovery and multistep purification, prior to the enzymatic/chemical digestion of GAGs, and finally their analysis. Moreover, 105–107 cells are usually required for compositional analysis. We report a sensitive, rapid, and quantitative analysis of GAGs present in a small number of cells. Commonly studied cell lines were selected based on phenotypic properties related to the biological functions of GAGs. These cells were lysed using a commercial surfactant reagent, sonicated, and digested with polysaccharide lyases. The resulting disaccharides were recovered by centrifugal filtration, labeled with 2-Aminoacridone, and analyzed by liquid chromatograp...
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Analysis of glycosaminoglycan-derived, precolumn, 2-Aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection
Nature Protocols, 2014Co-Authors: Nicola Volpi, Fabio Galeotti, Bo Yang, Robert J. LinhardtAbstract:Glycosaminoglycans (GAGs) possess considerable heterogeneity in average molecular mass, molecular mass range, disaccharide composition and content and position of sulfo groups. Despite recent technological advances in the analysis of GAGs, the determination of GAG disaccharide composition still remains challenging and provides key information required for understanding GAG function. Analysis of GAG-derived disaccharides relies on enzymatic treatment, providing one of the most practical and quantitative approaches for compositional mapping. Tagging the reducing end of disaccharides with an aromatic fluorescent label affords stable derivatives with properties that enable improved detection and resolution. HPLC with on-line electrospray ionization mass spectrometry (ESI-MS) offers a relatively soft ionization method for detection and characterization of sulfated oligosaccharides. GAGs obtained from tissues, biological fluids or cells are treated with various enzymes to obtain disaccharides that are fluorescently labeled with 2-Aminoacridone (AMAC) and resolved by different LC systems for high-sensitivity detection by fluorescence, and then they are unambiguously characterized by MS. The preparation and labeling of GAG-derived disaccharides can be performed in ∼1–2 d, and subsequent HPLC separation and on-line fluorescence detection and ESI-MS analysis takes another 1–2 h.
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analysis of glycosaminoglycan derived precolumn 2 aminoacridone labeled disaccharides with lc fluorescence and lc ms detection
Nature Protocols, 2014Co-Authors: Nicola Volpi, Fabio Galeotti, Bo Yang, Robert J. LinhardtAbstract:Analysis of glycosaminoglycan-derived, precolumn, 2-Aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection
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Analysis of glycosaminoglycan-derived, precolumn, 2-Aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection
Nature protocols, 2014Co-Authors: Nicola Volpi, Fabio Galeotti, Bo Yang, Robert J. LinhardtAbstract:Analysis of glycosaminoglycan-derived, precolumn, 2-Aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection
