The development of ToF-SIMS for in-situ glycosaminoglycan analysis
Glycosaminoglycans (GAGs) are linear polysaccharide chains composed of repeating disaccharide units with essential roles in several biological processes from embryonic patterning to modulation of blood vessel permeability. Despite their biological importance, GAG analysis is limited by a lack of ava...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2025
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| Online Access: | https://eprints.nottingham.ac.uk/80620/ |
| _version_ | 1848801259002265600 |
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| author | Milne, Lorna |
| author_facet | Milne, Lorna |
| author_sort | Milne, Lorna |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Glycosaminoglycans (GAGs) are linear polysaccharide chains composed of repeating disaccharide units with essential roles in several biological processes from embryonic patterning to modulation of blood vessel permeability. Despite their biological importance, GAG analysis is limited by a lack of available tools that can provide simultaneous spatial and compositional analysis. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a method of mass spectrometry imaging that enables the collection of compositional analysis through the generation of mass spectra, and spatial analysis through the conversion of spectra to an image. Previous utilisation of ToF-SIMS for in-situ GAG analysis has focussed on sulphate-containing ions that provide minimal compositional information.
In the approach presented here, a series of knockout cells lines mutant for components of the GAG biosynthetic pathway are analysed to generate and validate a library of SIMS ions that discriminate between different GAG types, including both sulphated and non-sulphated. These ions were acquired from biological samples that contain a milieu of biomolecules. Through the analysis of cell lines carrying knockouts of specific sulphotransferases, ions were identified that were discriminatory of both heparan sulphate (HS) and chondroitin sulphate (CS) sulphation modifications. To the best of our knowledge, this is the first example of the use of knockout cell lines to validate the likely biological origin of a SIMS ion.
Methods of multi variant analysis were optimised to enable the use of ions non-discriminatory of individual GAG types to act as an initial screening tool to identify GAG related changes in samples. Principal component analysis and partial least square regression discriminant analysis could use the intensity of such ions to discriminate between wild type (WT) and GAG synthetic mutant cell lines.
Ions identified in cellular samples were then applied to more complex tissue and used to investigate spatial and compositional changes in GAGs occurring in pathology. A particular focus was placed on understanding GAG related changes in renal tissue, including in diabetes, Alport syndrome and pregnancy. SIMS could identify the well-known loss of HS in diabetes and identified a previously unknown change in CS sulphation that occurs in WT pregnant mice but not Alport syndrome pregnant mice and could provide a potential mechanism for the exacerbated proteinuria observed during an Alport syndrome pregnancy. A workflow to use SIMS as a method of measuring endothelial glycocalyx (eGX) thickness was established to enable simultaneous spatial and compositional analysis of the carbohydrate rich layer, which is not possible using current techniques. Finally, high resolution SIMS images of human glomeruli were acquired. Again, to the best of our knowledge this is the first example of such analysis. A workflow was generated to identify GAG ion intensity changes over the individual layers of the glomerular filtration barrier to improve the understanding of where specifically within the barrier GAG related changes occur in pathology.
This thesis presents the development of ToF-SIMS as a method of in-situ GAG analysis that overcomes several limitations to enable label free, simultaneous spatial and compositional analysis. The technique is uniquely positioned to improve our understanding of fundamental GAG biology. SIMS can be used to investigate and identify relationships between different GAG types, including in-situ analysis of GAGs where their function remains poorly understood, such as in human embryonic development. Furthermore, SIMS can be used to discriminate the spatial distribution and composition of GAGs between healthy and disease state samples, including liquid and tissue biopsies, enabling improved understanding and detection of these complex molecules, potentially identifying new diagnostic and therapeutic targets. |
| first_indexed | 2025-11-14T21:04:36Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-80620 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T21:04:36Z |
| publishDate | 2025 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-806202025-08-20T15:26:33Z https://eprints.nottingham.ac.uk/80620/ The development of ToF-SIMS for in-situ glycosaminoglycan analysis Milne, Lorna Glycosaminoglycans (GAGs) are linear polysaccharide chains composed of repeating disaccharide units with essential roles in several biological processes from embryonic patterning to modulation of blood vessel permeability. Despite their biological importance, GAG analysis is limited by a lack of available tools that can provide simultaneous spatial and compositional analysis. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a method of mass spectrometry imaging that enables the collection of compositional analysis through the generation of mass spectra, and spatial analysis through the conversion of spectra to an image. Previous utilisation of ToF-SIMS for in-situ GAG analysis has focussed on sulphate-containing ions that provide minimal compositional information. In the approach presented here, a series of knockout cells lines mutant for components of the GAG biosynthetic pathway are analysed to generate and validate a library of SIMS ions that discriminate between different GAG types, including both sulphated and non-sulphated. These ions were acquired from biological samples that contain a milieu of biomolecules. Through the analysis of cell lines carrying knockouts of specific sulphotransferases, ions were identified that were discriminatory of both heparan sulphate (HS) and chondroitin sulphate (CS) sulphation modifications. To the best of our knowledge, this is the first example of the use of knockout cell lines to validate the likely biological origin of a SIMS ion. Methods of multi variant analysis were optimised to enable the use of ions non-discriminatory of individual GAG types to act as an initial screening tool to identify GAG related changes in samples. Principal component analysis and partial least square regression discriminant analysis could use the intensity of such ions to discriminate between wild type (WT) and GAG synthetic mutant cell lines. Ions identified in cellular samples were then applied to more complex tissue and used to investigate spatial and compositional changes in GAGs occurring in pathology. A particular focus was placed on understanding GAG related changes in renal tissue, including in diabetes, Alport syndrome and pregnancy. SIMS could identify the well-known loss of HS in diabetes and identified a previously unknown change in CS sulphation that occurs in WT pregnant mice but not Alport syndrome pregnant mice and could provide a potential mechanism for the exacerbated proteinuria observed during an Alport syndrome pregnancy. A workflow to use SIMS as a method of measuring endothelial glycocalyx (eGX) thickness was established to enable simultaneous spatial and compositional analysis of the carbohydrate rich layer, which is not possible using current techniques. Finally, high resolution SIMS images of human glomeruli were acquired. Again, to the best of our knowledge this is the first example of such analysis. A workflow was generated to identify GAG ion intensity changes over the individual layers of the glomerular filtration barrier to improve the understanding of where specifically within the barrier GAG related changes occur in pathology. This thesis presents the development of ToF-SIMS as a method of in-situ GAG analysis that overcomes several limitations to enable label free, simultaneous spatial and compositional analysis. The technique is uniquely positioned to improve our understanding of fundamental GAG biology. SIMS can be used to investigate and identify relationships between different GAG types, including in-situ analysis of GAGs where their function remains poorly understood, such as in human embryonic development. Furthermore, SIMS can be used to discriminate the spatial distribution and composition of GAGs between healthy and disease state samples, including liquid and tissue biopsies, enabling improved understanding and detection of these complex molecules, potentially identifying new diagnostic and therapeutic targets. 2025-07-30 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/80620/1/Lorna%20Milne_10169354_ThesisCorrections_FinalSubmission.pdf Milne, Lorna (2025) The development of ToF-SIMS for in-situ glycosaminoglycan analysis. PhD thesis, University of Nottingham. glycobiology glycosaminoglycans SIMS ToF-SIMS |
| spellingShingle | glycobiology glycosaminoglycans SIMS ToF-SIMS Milne, Lorna The development of ToF-SIMS for in-situ glycosaminoglycan analysis |
| title | The development of ToF-SIMS for in-situ glycosaminoglycan analysis |
| title_full | The development of ToF-SIMS for in-situ glycosaminoglycan analysis |
| title_fullStr | The development of ToF-SIMS for in-situ glycosaminoglycan analysis |
| title_full_unstemmed | The development of ToF-SIMS for in-situ glycosaminoglycan analysis |
| title_short | The development of ToF-SIMS for in-situ glycosaminoglycan analysis |
| title_sort | development of tof-sims for in-situ glycosaminoglycan analysis |
| topic | glycobiology glycosaminoglycans SIMS ToF-SIMS |
| url | https://eprints.nottingham.ac.uk/80620/ |