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J Charlwood

Publications and source records attributed to J Charlwood.

At least 19 recordsLinked to original sources
PubMed2001

Sialylation of human IgG-Fc carbohydrate by transfected rat alpha2,6-sialyltransferase.

A recombinant IgG3 antibody with Phe-243 replaced by Ala (FA243) was expressed in a CHO-K1 parental cell line. The resulting IgG-Fc-linked carbohydrate was significantly alpha2,3-sialylated (53% of glycans), as indicated by normal- and reverse-phase HPLC analyses. Following transfection of a rat alpha2,6-sialyltransferase gene into this parental cell line, IgG-Fc-linked glycans were sialylated (60% of glycans) such that the ratio of alpha2,6- to alpha2,3-linked sialic acid was 0.9:1.0. By comparison, the wild-type IgG3 (F243) is minimally sialylated (2-3% alpha2,3-linked), thus suggesting that sialylation is controlled primarily by the protein structure local to the carbohydrate and that the two sialyltransferases compete to sialylate the nascent oligosaccharide. The additional alpha2,6-sialylation affected the function of the recombinant antibody. FA243 IgG3 having both alpha2,6 and alpha2,3-sialylation restored recognition to wild-type IgG3 levels for human FcgammaRI, FcgammaRII, and target cell lysis by complement. We discuss how sialylation linkage could modulate IgG function.

Animals↗
PubMed2001

Characterization of the glycosylation profiles of Alzheimer's beta -secretase protein Asp-2 expressed in a variety of cell lines.

Amyloid 39-42 beta -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39-42 beta-peptide is formed from amyloid precursor protein by the sequential action of beta- and gamma-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have beta-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.

Alzheimer Disease↗
PubMed2001

Immobilisation of antibodies in gels allows the improved release and identification of glycans.

Human IgG and IgM, bovine IgM and three therapeutic IgG monoclonal antibodies have been separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Carbohydrates were then released from these immobilised proteins by direct enzymatic digestion, derivatised with a highly fluorescent probe and analysed by high performance liquid chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. This procedure not only allowed measurement of the purity of the intact antibodies but also provided detailed analysis of the complex mixtures of oligosaccharides covalently attached to these glycoproteins. The methodology out-lined allows the simultaneous processing of a number of glycoproteins separated on one single gel. In contrast to the release of carbohydrate from glycoproteins in solution, this procedure can also be conveniently applied when only impure glycoprotein is available.

Animals↗
PubMed2001

Facile sequencing of oligosaccharides by matrix-assisted laser desorption/ionisation on a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer.

N-Linked oligosaccharide mixtures released from a number of standard glycoproteins were derivatised with 3-acetylamino-6-acetylaminoacridine (AA-Ac) using reductive amination. Analysis of these mixtures using an experimental matrix-assisted laser desorption/ionisation (MALDI) hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer provided detailed information about the mass distribution of the glycan derivatives. Collision-induced dissociation of the singly protonated [M + H](+) ions also gave rise to a number of product ions produced by the sequential cleavage of the glycosidic linkages. As fragmentation of the positively charged species occurred predominantly in one direction, i.e., from the non-reducing end of the glycan to the AA-Ac moiety, a considerable amount of information could be obtained with ease about the sequence in which the sugar residues were attached to one another. This derivatisation procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from proteins separated by gel electrophoresis.

Carbohydrate Sequence↗
PubMed2001

Analysis of N-linked oligosaccharides: progress towards the characterisation of glycoprotein-linked carbohydrates.

The covalent attachment of carbohydrate to proteins is a very common co- or post-translational event in the biosynthesis of glycoproteins. The type and heterogeneity of these oligosaccharides can affect a range of physico-chemical and biological properties of a glycoprotein. Thus the development of sensitive, reliable and robust analytical methods for carbohydrate analysis is important in the pharmaceutical industry, especially in the recombinant production of experimental and therapeutic glycoproteins. In this report we have reviewed methodology for the in-gel enzymatic release of N-linked oligosaccharides from glycoproteins separated by electrophoresis. These oligosaccharides are derivatised by reductive amination using 3-acetamido-6-aminoacridine (AA-Ac), a novel, highly fluorescent probe. A major advantage of this technique is that glycan derivatives are amenable to analysis by an array of chromatographic and mass spectrometric methods, allowing the resolution and characterisation of a wide variety of glycan structures. It is hoped that in due course the methodology described will be applied to proteomics studies, especially in identifying the role of carbohydrate in protein function and disease.

Animals↗
PubMed2000

Analysis of N-linked oligosaccharides released from glycoproteins separated by two-dimensional gel electrophoresis.

Protocols have been developed for the characterization of carbohydrate covalently attached (N-linked) to an asparagine residue in glycoproteins, after separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Mixtures of proteins (each at a level from 0.5 to 50 microg) were resolved in the first dimension according to their isoelectric points (pI), followed by separation in the orthogonal axis on the basis of their molecular weights. Glycans were released directly from excised gel spots after digestion with PNGase F, with or without prior treatment with trypsin. In a third method, glycoproteins were electroblotted onto poly(vinylidene difluoride) before glycans were released by PNGase F. For all these procedures profiles of the neutral and sialic acid-containing oligosaccharide mixtures were obtained after derivatization with 3-acetamido-6-aminoacridine, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and/or high-performance liquid chromatography. Potential applications to proteomics are discussed.

Animals↗
PubMed2000

Early indication of effects of puromycin aminonucleoside using a fluorimetric assay of 2-aminoacridone-derivatized carbohydrates in urine.

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.

Aminoacridines↗
PubMed2000

Analysis of oligosaccharides by microbore high-performance liquid chromatography.

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.

Carbohydrate Sequence↗
PubMed2000

A probe for the versatile analysis and characterization of N-linked oligosaccharides.

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.

Carbohydrate Sequence↗
PubMed2000

Genotypes and phenotypes of patients in the UK with carbohydrate-deficient glycoprotein syndrome type 1.

18 UK patients (14 families) have been diagnosed with the carbohydrate-deficient glycoprotein syndrome (CDGS), type 1, on the basis of their clinical symptoms and/or abnormal electrophoretic patterns of serum transferrin. Eleven out of the 16 infants died before the age of 2 years. Patients from 12 families had a typical type 1 transferrin profile but one had a variant profile and another, who had many of the clinical features of CDGS type 1, had a normal profile. Eleven of the patients (10 families) with the typical type 1 profile had a deficiency of phosphomannomutase (PMM), (CDGS type 1a) but there was no correlation between residual enzyme activity and severity of disease. All these patients were compound heterozygotes for mutations in the phosphomannomutase (PMM2) gene, with 7 out of the 10 families having the common R141H mutation. Eight different mutations were found, including three novel ones. There was no correlation between genotype and phenotype, although siblings had similar phenotypes. Three patients, including the one with the normal transferrin profile, did not have a deficiency of phosphomannomutase or phosphomannose isomerase (CDGS 1b).

Cells, Cultured↗
PubMed2000

The image of the children's nurse: a study of the qualities required by families of children's nurses' uniform.

Traditional views of the nurse see 'her' in a nurse's uniform, whether she be a 'sex symbol', 'angel of mercy' or 'battle axe'. Children's nursing has, over recent years, moved away from the idea of the uniform into more child friendly clothes, though the traditional uniform is still presented to the public by the media. The staff members of a Children's Outpatients Department (COPD) of a large acute hospital requested knowledge of the qualities families required from the uniform of nurses caring for them, prior to any change in uniform. This study aimed to develop an understanding of these qualities via the use of a survey presented to those attending the COPD. The findings indicate that there is a complex relationship between the qualities that families wish the uniform to present. A balance is needed between presenting a professional authority figure, providing security for families and suggesting an approachable figure, to allow effective communication between staff and families.

Adult↗
PubMed1999

Carbohydrate release from picomole quantities of glycoprotein and characterisation of glycans by high-performance liquid chromatography and mass spectrometry.

Samples of 5 to 20 microg 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.

Amidohydrolases↗
PubMed1999

A detailed analysis of neutral and acidic carbohydrates in human milk.

Reverse- and normal-phase chromatography have been used to separate a number of standard human milk oligosaccharides derivatized via a reductive amination reaction with 2-aminoacridone (2-AMAC). Analytes were detected by spectrofluorimetry and injected simultaneously with a hydrolyzed dextran ladder derivatized with methyl-4-aminobenzoate. The latter probe does not fluoresce at the wavelength of emission by the 2-AMAC derivatives, and the derivatized, hydrolyzed dextran components were visualized by their ultraviolet absorbance. This procedure gave precise measurements of the "size" of 2-AMAC oligosaccharides in terms of their glucose equivalent values. Analytical amounts of 2-AMAC oligosaccharide standards were also isolated for further characterization by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry. MS-MS was also used to provide information on oligosaccharide sequences. This methodology was used successfully to characterize mixtures of neutral and acidic oligosaccharides from samples of human milk. This approach could be usefully applied to the study of glycoforms from a variety of samples such as those released from glycoproteins/glycolipids; these have been reported to be altered in a number of diseases, for example, cancer, cystic fibrosis, and autoimmune disease such as rheumatoid arthritis.

Aminoacridines↗
PubMed1999

Profiling of 2-aminoacridone derivatised glycans by electrospray ionization mass spectrometry.

Three different types of N-glycans, high-mannose, complex and hybrid, have been derivatised with 2-aminoacridone and analysed by electrospray mass spectrometry and tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, fitted with a nanoflow electrospray ion source. Relatively simple MS/MS fragmentation patterns have been observed for each type of glycan, allowing rapid elucidation of the order in which the monosaccharide residues making up these glycans are linked to one another.

Aminoacridines↗
PubMed1999

A chromatographic and mass spectrometric strategy for the analysis of oligosaccharides: determination of the glycan structures in porcine thyroglobulin.

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.

Aminoacridines↗
PubMed1999

Structural characterisation of N-linked glycan mixtures by precursor ion scanning and tandem mass spectrometric analysis

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.

Journal Article↗
PubMed1998

Two-dimensional chromatography in the analysis of complex glycans from transferrin.

Oligosaccharides released from human transferrin have been derivatized with 2-aminoacridone (2-AMAC) prior to analysis by either reversed- or normal-phase high-performance liquid chromatography. These separation methods are complementary and allow distinction between isomeric mono- and disialylated oligosaccharides, which terminate with Sia alpha 2-6Gal or Sia alpha 2-3Gal at the nonreducing end. Collected fractions of 2-AMAC-derivatized glycans were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, before and after desialylation.

Aminoacridines↗