Publications by authors named "Richard A Gardner"

21 Publications

  • Page 1 of 1

Interlaboratory evaluation of plasma N-glycan antennary fucosylation as a clinical biomarker for HNF1A-MODY using liquid chromatography methods.

Glycoconj J 2021 Mar 25. Epub 2021 Mar 25.

Ludger Ltd, Culham Science Centre, Oxfordshire, Abingdon, UK.

Antennary fucosylation alterations in plasma glycoproteins have been previously proposed and tested as a biomarker for differentiation of maturity onset diabetes of the young (MODY) patients carrying a functional mutation in the HNF1A gene. Here, we developed a novel LC-based workflow to analyze blood plasma N-glycan fucosylation in 320 diabetes cases with clinical features matching those at risk of HNF1A-MODY. Fucosylation levels measured in two independent research centers by using similar LC-based methods were correlated to evaluate the interlaboratory performance of the biomarker. The interlaboratory study showed good correlation between fucosylation levels measured for the 320 cases in the two centers with the correlation coefficient (r) of up to 0.88 for a single trait A3FG3S2. The improved chromatographic separation allowed the identification of six single glycan traits and a derived antennary fucosylation trait that were able to differentiate individuals carrying pathogenic mutations from benign or no HNF1A mutation cases, as determined by the area under the curve (AUC) of up to 0.94. The excellent (r = 0.88) interlaboratory performance of the glycan biomarker for HNF1A-MODY further supports the development of a clinically relevant diagnostic test measuring antennary fucosylation levels.
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http://dx.doi.org/10.1007/s10719-021-09992-wDOI Listing
March 2021

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin mediated clearance.

Haematologica 2021 Mar 25. Epub 2021 Mar 25.

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland; National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland; National Coagulation Centre, St James's Hospital, Dublin.

Terminal sialylation determines plasma VWF half-life. A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we show that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF:Ag levels (p.
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http://dx.doi.org/10.3324/haematol.2020.274720DOI Listing
March 2021

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes.

PLoS Negl Trop Dis 2021 Feb 2;15(2):e0009071. Epub 2021 Feb 2.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.
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http://dx.doi.org/10.1371/journal.pntd.0009071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880456PMC
February 2021

L1CAM as an E-selectin Ligand in Colon Cancer.

Int J Mol Sci 2020 Nov 5;21(21). Epub 2020 Nov 5.

Unidade de Ciências Biomoleculares Aplicadas (UCIBIO), Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLe) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 () coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLe in CRC. The SW620FUT6 cell line expressed high levels of sLe antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.
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http://dx.doi.org/10.3390/ijms21218286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672641PMC
November 2020

A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG.

Glycoconj J 2020 12 16;37(6):691-702. Epub 2020 Oct 16.

Ludger Ltd, Culham Science Centre, Abingdon, UK.

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson's r correlation coefficient of 0.8208 between the two methods.
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http://dx.doi.org/10.1007/s10719-020-09953-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679266PMC
December 2020

A Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Assay for the Relative Quantitation of Antennary Fucosylated -Glycans in Human Plasma.

Front Chem 2020 28;8:138. Epub 2020 Feb 28.

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands.

Changes in the abundance of antennary fucosylated glycans in human total plasma -glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. -linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of α2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer.
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http://dx.doi.org/10.3389/fchem.2020.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059190PMC
February 2020

Improved and semi-automated reductive β-elimination workflow for higher throughput protein O-glycosylation analysis.

PLoS One 2019 17;14(1):e0210759. Epub 2019 Jan 17.

Ludger Ltd, Culham Science Centre, Abingdon, Oxfordshire, United Kingdom.

Protein O-glycosylation has shown to be critical for a wide range of biological processes, resulting in an increased interest in studying the alterations in O-glycosylation patterns of biological samples as disease biomarkers as well as for patient stratification and personalized medicine. Given the complexity of O-glycans, often a large number of samples have to be analysed in order to obtain conclusive results. However, most of the O-glycan analysis work done so far has been performed using glycoanalytical technologies that would not be suitable for the analysis of large sample sets, mainly due to limitations in sample throughput and affordability of the methods. Here we report a largely automated system for O-glycan analysis. We adapted reductive β-elimination release of O-glycans to a 96-well plate system and transferred the protocol onto a liquid handling robot. The workflow includes O-glycan release, purification and derivatization through permethylation followed by MALDI-TOF-MS. The method has been validated according to the ICH Q2 (R1) guidelines for the validation of analytical procedures. The semi-automated reductive β-elimination system enabled for the characterization and relative quantitation of O-glycans from commercially available standards. Results of the semi-automated method were in good agreement with the conventional manual in-solution method while even outperforming it in terms of repeatability. Release of O-glycans for 96 samples was achieved within 2.5 hours, and the automated data acquisition on MALDI-TOF-MS took less than 1 minute per sample. This largely automated workflow for O-glycosylation analysis showed to produce rapid, accurate and reliable data, and has the potential to be applied for O-glycan characterization of biological samples, biopharmaceuticals as well as for biomarker discovery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210759PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336230PMC
October 2019

High-throughput Serum -Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes.

Mol Cell Proteomics 2019 01 21;18(1):3-15. Epub 2018 Sep 21.

From the ‡Center for Proteomics and Metabolomics.

-Glycosylation is a fundamentally important protein modification with a major impact on glycoprotein characteristics such as serum half-life and receptor interaction. More than half of the proteins in human serum are glycosylated, and the relative abundances of protein glycoforms often reflect alterations in health and disease. Several analytical methods are currently capable of analyzing the total serum -glycosylation in a high-throughput manner.Here we evaluate and compare the performance of three high-throughput released -glycome analysis methods. Included were hydrophilic-interaction ultra-high-performance liquid chromatography with fluorescence detection (HILIC-UHPLC-FLD) with 2-aminobenzamide labeling of the glycans, multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) with 8-aminopyrene-1,3,6-trisulfonic acid labeling, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with linkage-specific sialic acid esterification. All methods assessed the same panel of serum samples, which were obtained at multiple time points during the pregnancies and postpartum periods of healthy women and patients with rheumatoid arthritis (RA). We compared the analytical methods on their technical performance as well as on their ability to describe serum protein -glycosylation changes throughout pregnancy, with RA, and with RA disease activity.Overall, the methods proved to be similar in their detection and relative quantification of serum protein -glycosylation. However, the non-MS methods showed superior repeatability over MALDI-TOF-MS and allowed the best structural separation of low-complexity -glycans. MALDI-TOF-MS achieved the highest throughput and provided compositional information on higher-complexity -glycans. Consequentially, MALDI-TOF-MS could establish the linkage-specific sialylation differences within pregnancy and RA, whereas HILIC-UHPLC-FLD and xCGE-LIF demonstrated differences in α1,3- and α1,6-branch galactosylation. While the combination of methods proved to be the most beneficial for the analysis of total serum protein -glycosylation, informed method choices can be made for the glycosylation analysis of single proteins or samples of varying complexity.
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http://dx.doi.org/10.1074/mcp.RA117.000454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317482PMC
January 2019

Engineering and stable production of recombinant IgE for cancer immunotherapy and AllergoOncology.

J Allergy Clin Immunol 2018 04 31;141(4):1519-1523.e9. Epub 2018 Jan 31.

St John's Institute of Dermatology, School of Basic & Medical Biosciences, King's College London, London, United Kingdom; NIHR Biomedical Research Centre at Guy's and St Thomas's Hospitals and King's College London, London, United Kingdom; Breast Cancer Now Unit, School of Cancer & Pharmaceutical Sciences, King's College London, Guy's Cancer Centre, London, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.12.986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286379PMC
April 2018

Analysis of Three Epoetin Alpha Products by LC and LC-MS Indicates Differences in Glycosylation Critical Quality Attributes, Including Sialic Acid Content.

Anal Chem 2017 06 9;89(12):6455-6462. Epub 2017 Jun 9.

Reading School of Pharmacy, University of Reading , Reading, Berkshire RG6 6AP, United Kingdom.

Erythropoietin (EPO) is one of the main therapeutics used to treat anemic patients, greatly improving their quality of life. In this study, biosimilars Binocrit and a development product, called here CIGB-EPO, were compared to the originator product, Eprex. All three are epoetin alpha products, reputed to have similar glycosylation profiles. The quality, safety, and efficacy of this biotherapeutic depend on the following glycosylation critical quality attributes (GCQAs): sialylation, N-glycolyl-neuraminic acid (Neu5Gc) content, branching, N-acetyl-lactosamine (LacNAc) extensions, and O-acetylation pattern. Reverse-phase ultra-high-pressure liquid chromatography (RP-UHPLC) analysis of acid-released, 1,2-diamino-4,5-methylenedioxybenzene (DMB) labeled sialic acid derivatives and hydrophilic interaction liquid chromatography (HILIC) in combination with mass spectrometry (HILIC-UHPLC-MS) of procainamide (PROC) labeled N-glycans were the analytical tools used. An automated method for enzymatic release and PROC labeling was applied for the first time to the erythropoiesis stimulating agent (ESA) products, which facilitated novel, in-depth characterization, and allowed identification of precise structural features including the location of O-acetyl groups on sialic acid (SA) moieties. Samples were digested by a sialate-O-acetylesterase (NanS) to confirm the presence of O-acetyl groups. It was found that Eprex contained the greatest relative abundance of O-acetylated derivatives, Binocrit expressed the least Neu5Gc, and CIGB-EPO showed the greatest variety of high-mannose-phosphate structures. The sialylation and LacNAc extension patterns of the three ESAs were similar, with a maximum of four N-acetyl-neuraminic acid (Neu5Ac) moieties detected per glycan. Such differences in SA derivatization, particularly O-acetylation, could have consequences for the quality and safety of a biotherapeutic, as well as its efficacy.
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http://dx.doi.org/10.1021/acs.analchem.7b00353DOI Listing
June 2017

Characterization of a sialate-O-acetylesterase (NanS) from the oral pathogen Tannerella forsythia that enhances sialic acid release by NanH, its cognate sialidase.

Biochem J 2015 Dec 16;472(2):157-67. Epub 2015 Sep 16.

Integrated BioSciences, School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, U.K.

Tannerella forsythia, a Gram-negative member of the Bacteroidetes has evolved to harvest and utilize sialic acid. The most common sialic acid in humans is a mono-N-acetylated version termed Neu5Ac (5-N-acetyl-neuraminic acid). Many bacteria are known to access sialic acid using sialidase enzymes. However, in humans a high proportion of sialic acid contains a second acetyl group attached via an O-group, i.e. chiefly O-acetylated Neu5,9Ac2 or Neu5,4Ac2. This diacetylated sialic acid is not cleaved efficiently by many sialidases and in order to access diacetylated sialic acid, some organisms produce sialate-O-acetylesterases that catalyse the removal of the second acetyl group. In the present study, we performed bioinformatic and biochemical characterization of a putative sialate-O-acetylesterase from T. forsythia (NanS), which contains two putative SGNH-hydrolase domains related to sialate-O-acetylesterases from a range of organisms. Purification of recombinant NanS revealed an esterase that has activity against Neu5,9Ac2 and its glycolyl form Neu5Gc,9Ac. Importantly, the enzyme did not remove acetyl groups positioned at the 4-O position (Neu5,4Ac2). In addition NanS can act upon complex N-glycans released from a glycoprotein [erythropoietin (EPO)], bovine submaxillary mucin and oral epithelial cell-bound glycans. When incubated with its cognate sialidase, NanS increased sialic acid release from mucin and oral epithelial cell surfaces, implying that this esterase improves sialic acid harvesting for this pathogen and potentially other members of the oral microbiome. In summary, we have characterized a novel sialate-O-acetylesterase that contributes to the sialobiology of this important human pathogen and has potential applications in the analysis of sialic acid diacetylation of biologics in the pharmaceutical industry.
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http://dx.doi.org/10.1042/BJ20150388DOI Listing
December 2015

Changes to serum sample tube and processing methodology does not cause Intra-Individual [corrected] variation in automated whole serum N-glycan profiling in health and disease.

PLoS One 2015 1;10(4):e0123028. Epub 2015 Apr 1.

Ludger Ltd, Culham Science Centre, Oxford, Oxfordshire, OX14 3EB, United Kingdom.

Introduction: Serum N-glycans have been identified as putative biomarkers for numerous diseases. The impact of different serum sample tubes and processing methods on N-glycan analysis has received relatively little attention. This study aimed to determine the effect of different sample tubes and processing methods on the whole serum N-glycan profile in both health and disease. A secondary objective was to describe a robot automated N-glycan release, labeling and cleanup process for use in a biomarker discovery system.

Methods: 25 patients with active and quiescent inflammatory bowel disease and controls had three different serum sample tubes taken at the same draw. Two different processing methods were used for three types of tube (with and without gel-separation medium). Samples were randomised and processed in a blinded fashion. Whole serum N-glycan release, 2-aminobenzamide labeling and cleanup was automated using a Hamilton Microlab STARlet Liquid Handling robot. Samples were analysed using a hydrophilic interaction liquid chromatography/ethylene bridged hybrid(BEH) column on an ultra-high performance liquid chromatography instrument. Data were analysed quantitatively by pairwise correlation and hierarchical clustering using the area under each chromatogram peak. Qualitatively, a blinded assessor attempted to match chromatograms to each individual.

Results: There was small intra-individual variation in serum N-glycan profiles from samples collected using different sample processing methods. Intra-individual correlation coefficients were between 0.99 and 1. Unsupervised hierarchical clustering and principal coordinate analyses accurately matched samples from the same individual. Qualitative analysis demonstrated good chromatogram overlay and a blinded assessor was able to accurately match individuals based on chromatogram profile, regardless of disease status.

Conclusions: The three different serum sample tubes processed using the described methods cause minimal inter-individual variation in serum whole N-glycan profile when processed using an automated workstream. This has important implications for N-glycan biomarker discovery studies using different serum processing standard operating procedures.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123028PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382121PMC
February 2016

High-Throughput Analysis and Automation for Glycomics Studies.

Chromatographia 2015;78(5-6):321-333. Epub 2014 Nov 16.

Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands ; Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands.

This review covers advances in analytical technologies for high-throughput (HTP) glycomics. Our focus is on structural studies of glycoprotein glycosylation to support biopharmaceutical realization and the discovery of glycan biomarkers for human disease. For biopharmaceuticals, there is increasing use of glycomics in Quality by Design studies to help optimize glycan profiles of drugs with a view to improving their clinical performance. Glycomics is also used in comparability studies to ensure consistency of glycosylation both throughout product development and between biosimilars and innovator drugs. In clinical studies there is as well an expanding interest in the use of glycomics-for example in Genome Wide Association Studies-to follow changes in glycosylation patterns of biological tissues and fluids with the progress of certain diseases. These include cancers, neurodegenerative disorders and inflammatory conditions. Despite rising activity in this field, there are significant challenges in performing large scale glycomics studies. The requirement is accurate identification and quantitation of individual glycan structures. However, glycoconjugate samples are often very complex and heterogeneous and contain many diverse branched glycan structures. In this article we cover HTP sample preparation and derivatization methods, sample purification, robotization, optimized glycan profiling by UHPLC, MS and multiplexed CE, as well as hyphenated techniques and automated data analysis tools. Throughout, we summarize the advantages and challenges with each of these technologies. The issues considered include reliability of the methods for glycan identification and quantitation, sample throughput, labor intensity, and affordability for large sample numbers.
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http://dx.doi.org/10.1007/s10337-014-2803-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363487PMC
November 2014

Improved nonreductive O-glycan release by hydrazinolysis with ethylenediaminetetraacetic acid addition.

Anal Biochem 2014 May 5;453:29-37. Epub 2014 Mar 5.

Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands; Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands.

The study of protein O-glycosylation is receiving increasing attention in biological, medical, and biopharmaceutical research. Improved techniques are required to allow reproducible and quantitative analysis of O-glycans. An established approach for O-glycan analysis relies on their chemical release in high yield by hydrazinolysis, followed by fluorescent labeling at the reducing terminus and high-performance liquid chromatography (HPLC) profiling. However, an unwanted degradation known as "peeling" often compromises hydrazinolysis for O-glycan analysis. Here we addressed this problem using low-molarity solutions of ethylenediaminetetraacetic acid (EDTA) in hydrazine for O-glycan release. O-linked glycans from a range of different glycoproteins were analyzed, including bovine fetuin, bovine submaxillary gland mucin, and serum immunoglobulin A (IgA). The data for the O-glycans released by hydrazine with anhydrous EDTA or disodium salt dihydrate EDTA show high yields of the native O-glycans compared with the peeled product, resulting in a markedly increased robustness of the O-glycan profiling method. The presented method for O-glycan release demonstrates significant reduction in peeling and reduces the number of sample handling steps prior to release.
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http://dx.doi.org/10.1016/j.ab.2014.02.030DOI Listing
May 2014

Protein O-glycosylation analysis.

Biol Chem 2012 Aug;393(8):687-708

Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, P.O. Box 9600, NL-2300 RC Leiden, The Netherlands.

This review provides an overview on the methods available for analysis of O-glycosylation. Three major themes are addressed: analysis of released O-glycans including different O-glycan liberation, derivatization, and detection methods; analysis of formerly O-glycosylated peptides yielding information on O-glycan attachment sites; analysis of O-glycopeptides, representing by far the most informative but also most challenging approach for O-glycan analysis. Although there are various techniques available for the identification of O-linked oligosaccharides, the focus here is on MS fragmentation techniques such as collision-induced fragmentation, electron capture dissociation, and electron transfer dissociation. Finally, the O-glycan analytical challenges that need to be met will be discussed.
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http://dx.doi.org/10.1515/hsz-2012-0144DOI Listing
August 2012

Suppression of peeling during the release of O-glycans by hydrazinolysis.

Anal Biochem 2012 Apr 20;423(1):119-28. Epub 2012 Jan 20.

Ludger Ltd., Culham Science Centre, Oxfordshire OX14 3EB, UK.

The analysis of O-glycans is essential for better understanding their functions in biological processes. Although many techniques for O-glycan release have been developed, the hydrazinolysis release method is the best for producing O-glycans with free reducing termini in high yield. This release technique allows the glycans to be labeled with a fluorophore and analyzed by fluorescence detection. Under the hydrazinolysis release conditions, a side reaction is observed and causes the loss of monosaccharides from the reducing terminus of the glycans (known as peeling). Using bovine fetuin (because it contains the sialylated O-glycans most commonly found on biopharmaceuticals) and bovine submaxillary gland mucin (BSM), here we demonstrate that peeling can be greatly reduced when the sample is buffer exchanged prior to hydrazinolysis with solutions of either 0.1% trifluoroacetic acid (TFA) or low-molarity (100, 50, 20, and 5 mM) ethylenediaminetetraacetic acid (EDTA). The addition of calcium chloride to fetuin resulted in an increase in peeling, whereas subsequent washing with EDTA abolished this effect, suggesting a role of calcium and possibly other cations in causing peeling. The presented technique for sample preparation prior to hydrazinolysis greatly reduces the level of undesirable cleavage products in O-glycan analysis and increases the robustness of the method.
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http://dx.doi.org/10.1016/j.ab.2012.01.002DOI Listing
April 2012

Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate.

FEBS J 2007 Sep 22;274(18):4825-36. Epub 2007 Aug 22.

Department of Molecular Biology and Microbiology and Biomolecular Science Center, University of Central Florida, FL, USA.

Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments.
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http://dx.doi.org/10.1111/j.1742-4658.2007.06008.xDOI Listing
September 2007

Synthesis and biological evaluation of new acinetoferrin homologues for use as iron transport probes in mycobacteria.

J Med Chem 2004 Sep;47(20):4933-40

Department of Chemistry and Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, Florida 32816-2366, USA.

Four new acinetoferrin homologues were synthesized using a modular synthetic approach. Two linear and two cyclic imide derivatives were generated and evaluated for growth stimulating behavior in Mycobacterium avium subsp paratuberculosis. The yield for the tandem coupling of a functionalized aminohydroxamic acid motif (2 equiv) to a tert-butyl citrate derivative was significantly improved using DCC and N-hydroxysuccinimide. (1)H NMR spectroscopy (CD(3)OD) provided a convenient method for monitoring the final imidization step in TFA using the doublet patterns between 2.5 and 3.06 ppm. New protocols demonstrated that only a 20% growth enhancement was observed with M. avium subsp. paratuberculosis using the imide of acinetoferrin. Last, a siderophore from Streptomyces pilosus, deferrioxamine B, was shown to cross-feed M. avium subsp. paratuberculosis with the same efficiency as the more costly, native chelator, mycobactin J.
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http://dx.doi.org/10.1021/jm049805yDOI Listing
September 2004

The psychodynamics of patients with false memory syndrome (FMS).

J Am Acad Psychoanal Dyn Psychiatry 2004 ;32(1):77-90

Columbia University, College of Physicians & Surgeons, USA.

The False Memory Syndrome (FMS) diagnosis was very much in vogue from the late 1980s to the mid-1990s. It was an outgrowth of the belief held by many therapists that childhood sexual abuse was one of the most common causes of all forms of psychopathology. Although no longer in-vogue, the diagnosis is more recently being used for people who are falsely claiming that they were sexually abused by their priests. As was true in the earlier era, there were indeed many people who were sexually abused in childhood, but there were also many who were not and actually came to believe that they were, especially under the influence of therapists conducting "repressed memory therapy." Similarly, sexual abuse by priests has been a widespread phenomenon. Yet, there are still false accusers who are being led to believe by their overzealous therapists that they were indeed abused. As a psychoanalyst who has been asked to do forensic evaluations in many of these cases, I automatically ask myself questions about the psychodynamics of such falsely accusing patients. Here I describe those psychodynamic factors that I believe were operative in FMS cases, factors which in some cases apply to false sex abuse accusation against priests. Accordingly, this article is not simply of historical interest, but is still relevant and timely.
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http://dx.doi.org/10.1521/jaap.32.1.77.28336DOI Listing
August 2004

Interview criteria for assessing allegations of sexual abuse in children and adults.

J Am Acad Psychoanal Dyn Psychiatry 2003 ;31(2):297-323

Columbia University, College of Physicians and Surgeons, USA.

Freud's dilemma with regard to whether or not he should believe his patients' allegations of sexual abuse in childhood is well known. A century later, psychoanalysts are still dealing with this important issue. In this article, I describe the criteria I have developed in recent years, criteria that have been useful for me when attempting to determine whether my child and adult patients' allegations of sex abuse are more likely to be true or more likely to be false. Although these differentiating criteria were developed in the context of forensic evaluations, they should prove useful in clinical settings as well.
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http://dx.doi.org/10.1521/jaap.31.2.297.22112DOI Listing
November 2003