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Dive into the research topics where Louise Royle is active.

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Featured researches published by Louise Royle.


Analytical Biochemistry | 2008

HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software

Louise Royle; Matthew Campbell; Catherine M. Radcliffe; Dawn M. White; David J. Harvey; Jodie L. Abrahams; Yun-Gon Kim; George Henry; Nancy A. Shadick; Michael E. Weinblatt; David M. Lee; Pauline M. Rudd; Raymond A. Dwek

We present a robust, fully automatable technology platform that includes computer software for the detailed analysis of low femtomoles of N-linked sugars released from glycoproteins. Features include (i) sample immobilization in 96-well plates, glycan release, and fluorescent labeling; (ii) quantitative HPLC analysis, including monosaccharide sequence, linkage, and arm-specific information for charged and neutral glycans; (iii) automatic structural assignment of peaks from HPLC profiles via web-based software that accesses our database (GlycoBase) of more than 350 N-glycan structures, including 117 present in the human serum glycome; and (iv) software (autoGU) that progressively analyzes data from exoglycosidase digestions to produce a refined list of final structures. The N-glycans from a plate of 96 samples can be released and purified in 2 or 3 days and profiled in 2 days. This strategy can be used for (i) identification and screening of disease biomarkers and (ii) monitoring the production of therapeutic glycoproteins, allowing optimization of production conditions. This technology is also suitable for preparing released glycans for other analytical techniques. Here we demonstrate its application to rheumatoid arthritis using 5 microl of patient serum.


Proteomics | 2009

Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds

David J. Harvey; Anthony H. Merry; Louise Royle; Matthew Campbell; Raymond A. Dwek; Pauline M. Rudd

Symbolic diagrams are commonly used to depict N‐ and O‐linked glycans but there is no general consensus as to how individual constituent monosaccharides or linkages are shown. This article proposes a system that avoids ambiguities inherent in most other systems and is appropriate for both hand drawing and computer applications. Constituent monosaccharides are depicted by shapes modified to show OAc, deoxy, etc. Linkage is indicated by the bond angle and anomericity by solid (β) or dashed (α) lines.


Bioinformatics | 2008

GlycoBase and autoGU

Matthew Campbell; Louise Royle; Catherine M. Radcliffe; Raymond A. Dwek; Pauline M. Rudd

SUMMARY The development of robust high-performance liquid chromatography (HPLC) technologies continues to improve the detailed analysis and sequencing of glycan structures released from glycoproteins. Here, we present a database (GlycoBase) and analytical tool (autoGU) to assist the interpretation and assignment of HPLC-glycan profiles. GlycoBase is a relational database which contains the HPLC elution positions for over 350 2-AB labelled N-glycan structures together with predicted products of exoglycosidase digestions. AutoGU assigns provisional structures to each integrated HPLC peak and, when used in combination with exoglycosidase digestions, progressively assigns each structure automatically based on the footprint data. These tools are potentially very promising and facilitate basic research as well as the quantitative high-throughput analysis of low concentrations of glycans released from glycoproteins. AVAILABILITY http://glycobase.ucd.ie


Methods of Molecular Biology | 2006

Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions.

Louise Royle; Catherine M. Radcliffe; Raymond A. Dwek; Pauline M. Rudd

In contrast to the linear sequences of protein and DNA, oligosaccharides are branched structures. In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants. Glycan analysis therefore requires high-resolution separation techniques that can provide detailed structural analysis, including both monosaccharide sequence and linkage information. This chapter describes how a combination of high-performance liquid chromatography (HPLC) and exoglycosidase enzyme array digestions can deliver quantitative glycan analysis of sugars released from glycoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel bands by matching HPLC elution positions with a database of standard glycans.


Glycobiology | 2008

A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression

Umi M Abd Hamid; Louise Royle; Radka Saldova; Catherine M. Radcliffe; David J. Harvey; Sarah J. Storr; María Pardo; Robin Antrobus; Caroline Chapman; Nicole Zitzmann; J.F.R. Robertson; Raymond A. Dwek; Pauline M. Rudd

Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression.


Analytical Biochemistry | 2008

Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry.

David J. Harvey; Louise Royle; Catherine M. Radcliffe; Pauline M. Rudd; Raymond A. Dwek

Negative ion electrospray (ESI) fragmentation spectra derived from anion-adducted glycans were evaluated for structural determination of N-linked glycans and found to be among the most useful mass spectrometric techniques yet developed for this purpose. In contrast to the more commonly used positive ion spectra that contain isobaric ions formed by losses from different regions of the molecules and often lead to ambiguous deductions, the negative ion spectra contain ions that directly reflect structural features such as the branching pattern, location of fucose, and the presence of bisecting GlcNAc. These structural features are sometimes difficult to determine by traditional methods. Furthermore, the spectra give structural information from mixtures of isomers and from single compounds. The method was evaluated with well-characterized glycans from IgG and used to explore structures of N-linked glycans released from serum glycoproteins with the aim of identifying biomarkers for cancer. Quantities of glycans were measured by ESI and by matrix-assisted laser desorption ionization mass spectrometry; each technique produced virtually identical results for the neutral desialylated glycans.


Journal of Biological Chemistry | 2006

The Hemopexin and O-Glycosylated Domains Tune Gelatinase B/MMP-9 Bioavailability via Inhibition and Binding to Cargo Receptors

Philippe E. Van den Steen; Ilse Van Aelst; Vibeke Hvidberg; Helene Piccard; Pierre Fiten; Christian Jacobsen; Søren K. Moestrup; Simon Fry; Louise Royle; Mark R. Wormald; Russell Wallis; Pauline M. Rudd; Raymond A. Dwek; Ghislain Opdenakker

Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of ∼64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a compact three-dimensional structure. The OG and hemopexin domains have no influence on the cleavage efficiency of MMP-9 substrates. In contrast, the hemopexin domain contains a binding site for the cargo receptor low density lipoprotein receptor-related protein-1 (LRP-1). Furthermore, megalin/LRP-2 is identified as a new functional receptor for the hemopexin domain of MMP-9, able to mediate the endocytosis and catabolism of the enzyme. The OG domain is required to correctly orient the hemopexin domain for inhibition by TIMP-1 and internalization by LRP-1 and megalin. Therefore, the OG and hemopexin domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs.


Proteomics | 2001

A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands.

Pauline M. Rudd; Cristina Colominas; Louise Royle; Neil Murphy; Edmund Hart; Anthony H. Merry; Holger Hebestreit; Raymond A. Dwek

The majority of biologically active proteins are glycosylated, therefore any approach to proteomics which fails to address the analysis of oligosaccharides is necessarily incomplete. To appreciate the structure of a glycoprotein fully, to understand the roles for the attached oligosaccharides and to monitor disease associated changes it is necessary to visualise the sugars as well as the protein. To achieve this aim when biological samples are available at the low microgram level or less has involved increasing the sensitivity of the technology for glycan analysis. Since one protein may have many different oligosaccharides attached to it (glycoforms) this is a major technical challenge. CD59, for example, has over 100 different sugars at one N‐linked glycosylation site. Applications of recently developed technology suggest that it is now becoming realistic to extend the proteomics analysis of glycoproteins to include details of glycosylation. This is achieved by releasing the N‐glycans from the protein in a gel by optimised peptide‐N‐glycosidase F digestion. The released glycans are then tagged with the fluorophore, 2‐amino benzamide. The labelled glycan pools (containing 50–100 femtomoles of glycans) are resolved by predictive normal phase high performance liquid chromatography (HPLC) on an amide based column or by reverse phase HPLC on a C18 column. Preliminary structural assignments are confirmed by exoglycosidase array digestions of the entire glycan pool. Complementary matrix‐assisted laser desorption/ionization‐mass spectrometry, which requires 10–20 times as much sugar for a single run, can be used where there is sufficient material. This provides a composition analysis but not linkage information.


Glycobiology | 2008

The O-linked glycosylation of secretory/shed MUC1 from an advanced breast cancer patient's serum

Sarah J. Storr; Louise Royle; Caroline Chapman; Umi M Abd Hamid; J.F.R. Robertson; Andrea Murray; Raymond A. Dwek; Pauline M. Rudd

MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-linked glycosylation of MUC1 in cancer has been implicated in disease progression. We investigated the O-linked glycosylation of MUC1 purified from the serum of an advanced breast cancer patient. O-Glycans were released by hydrazinolysis and analyzed by liquid chromatography-electrospray ionization-mass spectrometry and by high performance liquid chromatography coupled with sequential exoglycosidase digestions. Core 1 type glycans (83%) dominated the profile which also confirmed high levels of sialylation: 80% of the glycans were mono-, di- or trisialylated. Core 2 type structures contributed approximately 17% of the assigned glycans and the oncofoetal Thomsen-Friedenreich (TF) antigen (Galbeta1-3GalNAc) accounted for 14% of the total glycans. Interestingly, two core 1 type glycans were identified that had sialic acid alpha2-8 linked to sialylated core 1 type structures (9% of the total glycan pool). This is the first O-glycan analysis of MUC1 from the serum of a breast cancer patient; the results suggest that amongst the cell lines commonly used to express recombinant MUC1 the T47D cell line processes glycans that are most similar to patient-derived material.


Cell Reports | 2016

Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

Anna Janina Behrens; Snezana Vasiljevic; Laura K. Pritchard; David J. Harvey; Rajinder S. Andev; Stefanie A. Krumm; Weston B. Struwe; Albert Cupo; Abhinav Kumar; Nicole Zitzmann; Gemma E. Seabright; Holger B. Kramer; Daniel Spencer; Louise Royle; Jeong Hyun Lee; P. J. Klasse; Dennis R. Burton; Ian A. Wilson; Andrew B. Ward; Rogier W. Sanders; John P. Moore; Katie J. Doores; Max Crispin

Summary The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

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Daniel Spencer

University of Southampton

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