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Comprehensive stereochemical sequencing of carbohydrates and characterisation oftheir binding partners using hyphenated mass spectrometry methods
[Thesis]. Manchester, UK: The University of Manchester; 2017.
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Abstract
Glycans and their conjugates form the largest and most diverse class of biologicalmolecules found within nature. These glycosides are vital for numerous cellular functionsincluding recognition events, protein stabilisation and energy storage, to name a few.Additionally, abnormalities within these structures are associated with a wide range ofdisease states. As a result, robust analytical techniques capable of in depth characterisationof carbohydrates and their binding partners are required. Currently, liquid chromatographycoupled with tandem mass spectrometry (MS2) is the ‘gold standard’ for characterisingthese species. However there are inherent challenges for ‘sequencing’ carbohydrates giventhat most structures are diastereomeric. As a result MS alone is insufficient to fullyelucidate all stereochemical and often regiochemical information and alternative analyticaltechniques have inherent issues meaning that they are not suitable for medium/highthroughputanalysis. To facilitate elucidation of these structures, ion mobility spectrometry(IMS) has been used in-line with MS2. IMS of mono- and di-saccharide product ionsgenerate by collision-induced dissociation (CID) of various glycans and their conjugatesenables unambiguous identification of the monomer and the regio-/stereo-chemistry of theglycosidic bond, independent of the precursor structure.Also, given the prominence of glycans in biological recognition events, high-throughputtechniques capable of elucidating and characterising carbohydrate to glycan-bindingprotein (GBP) interactions are highly sought after. Historically, (micro)array strategies areemployed to screen large numbers of biological interactions, with detection conventionallyachieved with fluorescent tagging. The major disadvantage of this approach is therequirement of a labelling step to facilitate detection of glycan-GBP binding. MS offers theability to unambiguously identify GBPs when combined with routine bottom-upproteomics strategies, namely on-chip proteolysis followed by mass fingerprinting andMS2 analysis and subsequent comparison to protein databases.It is anticipated that these methodologies developed throughout these studies, both forcarbohydrate sequencing and the characterisation of glycan-binding proteins, will greatlyadd to the Glycomics toolbox.