Award details

Advanced glycosaminoglycan sequencing

Reference	BB/L023717/1
Principal Investigator / Supervisor	Dr Mark Skidmore
Co-Investigators / Co-Supervisors
Institution	Keele University
Department	Inst for Science and Tech in Medicine
Funding type	Research
Value (£)	330,456
Status	Completed
Type	Research Grant
Start date	28/11/2014
End date	30/11/2018
Duration	48 months

Abstract

The sequence determination of the biologically important glycosaminoglycan (GAG) polysaccharides remains a significant technical challenge for several reasons. These include a difficulty in obtaining sufficient quanitties of purified GAGs from biological samples, problematic labelling chemistries, insensitive instrumentation and the reliance on rare, and thus expensive, exoglycosidase enzymes to elucidate sequence identity. This project will develop, evaluate and validate a novel approach in which two fundamental and radical improvements are incorporated. These are to combine a recently developed approach for efficient tethering of GAG saccharides via their non-reducing ends together with a vastly improved fluorescent label (BODIPY) and state-of-the-art detection system (LIF), developed in collaboration with an Industrial partner (MI Engineering Ltd.). This permits a novel "dual-end" sequencing method to be developed, permitting access to sequence information from both ends of the molecule, and providing two complementary opportunities to obtain sequence information. This improved sequencing strategy with vastly improved detection (at least 20,000 fold, accessing zeptomole levels for the first time) will provide a powerful sequencing tool for the high throughput analysis of biologically and medically important GAGs, and open up new opportunities to exploit GAGs in biomedical applications.

Summary

The interaction between proteins and cell-surface ligands underpin many biological processes such as cell growth, homeostasis, apoptosis and the ability of pathogens to invade host cells. One important family of cell surface ligands are the carbohydrate family of glycosaminoglycans (GAGs) found on the surface of almost all mammalian cells. Analysis of the sequence of the linear block-like arrangement of these GAGs is a significant technical challenge due to only miniscule quantities of pure material being (readily) available, poor chemistries and insensitive detection equipment. This research aims to develop and test a new method for sequencing these GAG polysaccharides utilising recent fundamental improvements made by the applicant. These developments include the exploitation of the opposite end of the molecule than is conventionally used (non-reducing end), with a vastly improved labelling mechanism and an advanced detection system for the conventional end of the molecule (reducing end). This approach will provide a powerful and sensitive sequencing technique capable of employing both ends of the GAG saccharide for characterization. The information gained from GAG sequencing will enable researchers to study previously elusive structures and processes that are biologically and medically significant.

Impact Summary

The biologically and medically family of carbohydrates, the glycosaminoglycans (GAGs), are present on the cell surface of almost all mammalian cells and are implicated in many crucial biological mechanisms such as cell growth, division, homeostasis and pathogen invasion. However, analysis of their sequence is a significant technical challenge compounded by the difficulty in obtaining sufficient purified material from biological samples. Sequencing techniques to date rely on relatively large amounts of starting material and are generally difficult, labour intensive, time consuming and relatively insensitive. The successful development of a rapid, sensitive and high throughput sequencing strategy will permit rapid progress to be made on investigations into the structure:function relationships of GAG saccharides and will ultimately contribute significantly to our understanding of GAG polysaccharides as major components of the glycome. This is a crucial facet of post-genome science, and will open up major new exploitation.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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