Publications by authors named "Radoslaw P Kozak"

16 Publications

  • Page 1 of 1

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

Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis.

Sci Rep 2020 07 31;10(1):12903. Epub 2020 Jul 31.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK.

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host's immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with ManGlcNAc being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.
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http://dx.doi.org/10.1038/s41598-020-69753-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395719PMC
July 2020

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Authors:
Maria Lorna A De Leoz David L Duewer Adam Fung Lily Liu Hoi Kei Yau Oscar Potter Gregory O Staples Kenichiro Furuki Ruth Frenkel Yunli Hu Zoran Sosic Peiqing Zhang Friedrich Altmann Clemens Grunwald-Grube Chun Shao Joseph Zaia Waltraud Evers Stuart Pengelley Detlev Suckau Anja Wiechmann Anja Resemann Wolfgang Jabs Alain Beck John W Froehlich Chuncui Huang Yan Li Yaming Liu Shiwei Sun Yaojun Wang Youngsuk Seo Hyun Joo An Niels-Christian Reichardt Juan Echevarria Ruiz Stephanie Archer-Hartmann Parastoo Azadi Len Bell Zsuzsanna Lakos Yanming An John F Cipollo Maja Pucic-Bakovic Jerko Štambuk Gordan Lauc Xu Li Peng George Wang Andreas Bock René Hennig Erdmann Rapp Marybeth Creskey Terry D Cyr Miyako Nakano Taiki Sugiyama Pui-King Amy Leung Paweł Link-Lenczowski Jolanta Jaworek Shuang Yang Hui Zhang Tim Kelly Song Klapoetke Rui Cao Jin Young Kim Hyun Kyoung Lee Ju Yeon Lee Jong Shin Yoo Sa-Rang Kim Soo-Kyung Suh Noortje de Haan David Falck Guinevere S M Lageveen-Kammeijer Manfred Wuhrer Robert J Emery Radoslaw P Kozak Li Phing Liew Louise Royle Paulina A Urbanowicz Nicolle H Packer Xiaomin Song Arun Everest-Dass Erika Lattová Samanta Cajic Kathirvel Alagesan Daniel Kolarich Toyin Kasali Viv Lindo Yuetian Chen Kudrat Goswami Brian Gau Ravi Amunugama Richard Jones Corné J M Stroop Koichi Kato Hirokazu Yagi Sachiko Kondo C T Yuen Akira Harazono Xiaofeng Shi Paula E Magnelli Brian T Kasper Lara Mahal David J Harvey Roisin O'Flaherty Pauline M Rudd Radka Saldova Elizabeth S Hecht David C Muddiman Jichao Kang Prachi Bhoskar Daniele Menard Andrew Saati Christine Merle Steven Mast Sam Tep Jennie Truong Takashi Nishikaze Sadanori Sekiya Aaron Shafer Sohei Funaoka Masaaki Toyoda Peter de Vreugd Cassie Caron Pralima Pradhan Niclas Chiang Tan Yehia Mechref Sachin Patil Jeffrey S Rohrer Ranjan Chakrabarti Disha Dadke Mohammedazam Lahori Chunxia Zou Christopher Cairo Béla Reiz Randy M Whittal Carlito B Lebrilla Lauren Wu Andras Guttman Marton Szigeti Benjamin G Kremkow Kelvin H Lee Carina Sihlbom Barbara Adamczyk Chunsheng Jin Niclas G Karlsson Jessica Örnros Göran Larson Jonas Nilsson Bernd Meyer Alena Wiegandt Emy Komatsu Helene Perreault Edward D Bodnar Nassur Said Yannis-Nicolas Francois Emmanuelle Leize-Wagner Sandra Maier Anne Zeck Albert J R Heck Yang Yang Rob Haselberg Ying Qing Yu William Alley Joseph W Leone Hua Yuan Stephen E Stein

Mol Cell Proteomics 2020 01 7;19(1):11-30. Epub 2019 Oct 7.

Mass Spectrometry Data Center, Biomolecular Measurement Division, Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Drive Gaithersburg, Maryland 20899.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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http://dx.doi.org/10.1074/mcp.RA119.001677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944243PMC
January 2020

Method comparison for N-glycan profiling: Towards the standardization of glycoanalytical technologies for cell line analysis.

PLoS One 2019 7;14(10):e0223270. Epub 2019 Oct 7.

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

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223270PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779296PMC
March 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

Variation of Human Salivary O-Glycome.

PLoS One 2016 9;11(9):e0162824. Epub 2016 Sep 9.

Centre for Proteomics and Metabolomics Leiden University Medical Centre, Leiden, The Netherlands.

The study of saliva O-glycosylation is receiving increasing attention due to the potential of glycans for disease biomarkers, but also due to easy access and non-invasive collection of saliva as biological fluid. Saliva is rich in glycoproteins which are secreted from the bloodstream or produced by salivary glands. Mucins, which are highly O-glycosylated proteins, are particularly abundant in human saliva. Their glycosylation is associated with blood group and secretor status, and represents a reservoir of potential disease biomarkers. This study aims to analyse and compare O-glycans released from whole human mouth saliva collected 3 times a day from a healthy individual over a 5 days period. O-linked glycans were released by hydrazinolysis, labelled with procainamide and analysed by ultra-high performance liquid chromatography with fluorescence detection (UHPLC-FLR) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). The sample preparation method showed excellent reproducibility and can therefore be used for biomarker discovery. Our data demonstrates that the O-glycosylation in human saliva changes significantly during the day. These changes may be related to changes in the salivary concentrations of specific proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162824PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017618PMC
August 2017

Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS.

Anal Chem 2016 09 15;88(17):8562-9. Epub 2016 Aug 15.

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

Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.
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http://dx.doi.org/10.1021/acs.analchem.6b01639DOI Listing
September 2016

Longitudinal monitoring of immunoglobulin A glycosylation during pregnancy by simultaneous MALDI-FTICR-MS analysis of N- and O-glycopeptides.

Sci Rep 2016 06 15;6:27955. Epub 2016 Jun 15.

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

Immunoglobulin A (IgA) is a glycoprotein of which altered glycosylation has been associated with several pathologies. Conventional methods for IgA N- and O-glycosylation analysis are tedious, thus limiting such analyses to small sample sizes. Here we present a high-throughput strategy for the simultaneous analysis of serum-derived IgA1 N- and O-glycopeptides using matrix-assisted laser/desorption ionisation Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry (MS). Six non-fucosylated diantennary complex type glycoforms were detected on the Asn144-containing glycopeptide. Thirteen distinct glycoforms were identified for the Asn340-containing tailpiece glycopeptide, mainly of the diantennary complex type, and low amounts of triantennary glycoforms. Simultaneously with these N-glycopeptides, 53 compositional glycoforms of the hinge region O-glycopeptide were profiled in a single high resolution MALDI-FTICR spectrum. Since many pregnancy associated changes have been recognized for immunoglobulin G, we sought to demonstrate the clinical applicability of this method in a cohort of 29 pregnant women, from whom samples were collected at three time points during pregnancy and three time points after delivery. Pregnancy associated changes of N-glycan bisection were different for IgA1 as compared to IgG-Fc described earlier. We foresee further applications of the developed method for larger patient cohorts to study IgA N- and O-glycosylation changes in pathologies.
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http://dx.doi.org/10.1038/srep27955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908400PMC
June 2016

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

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization-mass spectrometry.

Anal Biochem 2015 Oct 12;486:38-40. Epub 2015 Jun 12.

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

One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography-(ultra-)high-performance liquid chromatography [HILIC-(U)HPLC]. Here, we compare the data obtained by (U)HPLC-fluorescence (FLR) coupled to electrospray ionization-mass spectrometry (ESI-MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.
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http://dx.doi.org/10.1016/j.ab.2015.06.006DOI Listing
October 2015

Development of a Schistosoma mansoni shotgun O-glycan microarray and application to the discovery of new antigenic schistosome glycan motifs.

Int J Parasitol 2015 Jun 26;45(7):465-75. Epub 2015 Mar 26.

Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.

Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets.
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http://dx.doi.org/10.1016/j.ijpara.2015.02.008DOI Listing
June 2015

Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a mucin O-glycome designer array.

J Biol Chem 2014 Jun 21;289(23):16462-77. Epub 2014 Apr 21.

From the Glycosciences Laboratory, Department of Medicine, Imperial College London, W12 0NN London, United Kingdom,

Monoclonal antibody F77 was previously raised against human prostate cancer cells and has been shown to recognize a carbohydrate antigen, but the carbohydrate sequence of the antigen was elusive. Here, we make multifaceted approaches to characterize F77 antigen, including binding analyses with the glycolipid extract of the prostate cancer cell line PC3, microarrays with sequence-defined glycan probes, and designer arrays from the O-glycome of an antigen-positive mucin, in conjunction with mass spectrometry. Our results reveal F77 antigen to be expressed on blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone. We show that mAb F77 can also bind to blood group A and B analogs but with lower intensities. We propose that the close association of F77 antigen with prostate cancers is a consequence of increased blood group H expression together with up-regulated branching enzymes. This is in contrast to other epithelial cancers that have up-regulated branching enzymes but diminished expression of H antigen. With knowledge of the structure and prevalence of F77 antigen in prostate cancer, the way is open to explore rationally its application as a biomarker to detect F77-positive circulating prostate cancer-derived glycoproteins and tumor cells.
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http://dx.doi.org/10.1074/jbc.M114.558932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047413PMC
June 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

Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis.

Brain 2013 Dec 7;136(Pt 12):3618-24. Epub 2013 Oct 7.

1 Institute of Biomedical and Clinical Science, University of Exeter Medical School, St. Luke's Campus, Heavitree Road, EX1 2LU, Exeter, Devon, UK.

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.
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http://dx.doi.org/10.1093/brain/awt270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859217PMC
December 2013

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