Studying protein interaction and glycosylation with chemical proteomics
<p>Protein glycosylation is present in all kingdoms of life, from small virus particles to complex living organisms. It is the most abundant and diverse form of post-translational modification, ranging from the attachment of a single monosaccharide to entire sugar chains onto cellular proteins...
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| Format: | Thesis |
| Language: | English |
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2020
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| _version_ | 1817931838365106176 |
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| author | Nadal, S |
| author2 | Davis, B |
| author_facet | Davis, B Nadal, S |
| author_sort | Nadal, S |
| collection | OXFORD |
| description | <p>Protein glycosylation is present in all kingdoms of life, from small virus particles to complex living organisms. It is the most abundant and diverse form of post-translational modification, ranging from the attachment of a single monosaccharide to entire sugar chains onto cellular proteins. This modification affects the recognition and interaction of proteins with their interaction partners, altering their role in health and diseases. Despite its importance, the study of protein glycosylation has lacked behind other biomolecules and post-translational modifications, partly because of absence of available tools and methods. This doctoral thesis aims at expanding existing analytical approaches and developing new technologies to study the different types of protein glycosylation using tandem mass spectrometry.</p>
<p>The second chapter demonstrates de novo determination of the sequence and protein N-glycosylation of the anti- H2AX monoclonal antibody, enabling structural modelling of the antigen recognition and laying the groundwork for further development of this diagnostic reagent for DNA damage in cancers. Chapter 3 focusses on improving analysis of protein O-glycosylation using tandem mass spectrometry. The use of low-energy stepped collision energy (SCE) methods is described for improved characterisation of the glycan part and site-localisation of protein O-glycosylation. SCE-HCD methods such as 10-20% of normalised collision energy (NCE) show the formation of valuable fragment ions enabling a more accurate identification of O-glycopeptides.</p>
<p>The O-GlcNAc modification is a particular form of O-glycosylation, consisting in the attachment of a single N-acetylglucosamine sugar to proteins. In chapter 4, the role of O-GlcNAc as an epigenetic mark on H2B-S36 is investigated. Homogeneously GlcNAcylated histones and nucleosomes were synthesised and used in pull-down ex- periments to enrich putative interaction partners for the H2B-S36-GlcNAc modifica- tion. Mass spectrometry-based quantitative interaction proteomics revealed a direct interaction between H2B-S36-O-GlcNAcylated nucleosomes and the TCF20 transcrip- tion factor, suggesting a possible signalling role for the O-GlcNAc modification. Lastly, chapter 5 shows that synthetic histones with reactive side chains can be used to co- valently trap interaction partners in a native context, identifying their binding site by crosslinking mass spectrometry with amino-acid resolution inside the interaction.</p> |
| first_indexed | 2024-03-07T07:04:09Z |
| format | Thesis |
| id | oxford-uuid:09dd50bc-772d-49cc-a06f-53c86a33710f |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:28:23Z |
| publishDate | 2020 |
| record_format | dspace |
| spelling | oxford-uuid:09dd50bc-772d-49cc-a06f-53c86a33710f2024-12-01T11:11:05ZStudying protein interaction and glycosylation with chemical proteomicsThesishttp://purl.org/coar/resource_type/c_db06uuid:09dd50bc-772d-49cc-a06f-53c86a33710fProteomicsBiochemistryBioorganic chemistryPost-translational modificationEnglishHyrax Deposit2020Nadal, SDavis, BMohammed, SBenesch, JHeck, AJR<p>Protein glycosylation is present in all kingdoms of life, from small virus particles to complex living organisms. It is the most abundant and diverse form of post-translational modification, ranging from the attachment of a single monosaccharide to entire sugar chains onto cellular proteins. This modification affects the recognition and interaction of proteins with their interaction partners, altering their role in health and diseases. Despite its importance, the study of protein glycosylation has lacked behind other biomolecules and post-translational modifications, partly because of absence of available tools and methods. This doctoral thesis aims at expanding existing analytical approaches and developing new technologies to study the different types of protein glycosylation using tandem mass spectrometry.</p> <p>The second chapter demonstrates de novo determination of the sequence and protein N-glycosylation of the anti- H2AX monoclonal antibody, enabling structural modelling of the antigen recognition and laying the groundwork for further development of this diagnostic reagent for DNA damage in cancers. Chapter 3 focusses on improving analysis of protein O-glycosylation using tandem mass spectrometry. The use of low-energy stepped collision energy (SCE) methods is described for improved characterisation of the glycan part and site-localisation of protein O-glycosylation. SCE-HCD methods such as 10-20% of normalised collision energy (NCE) show the formation of valuable fragment ions enabling a more accurate identification of O-glycopeptides.</p> <p>The O-GlcNAc modification is a particular form of O-glycosylation, consisting in the attachment of a single N-acetylglucosamine sugar to proteins. In chapter 4, the role of O-GlcNAc as an epigenetic mark on H2B-S36 is investigated. Homogeneously GlcNAcylated histones and nucleosomes were synthesised and used in pull-down ex- periments to enrich putative interaction partners for the H2B-S36-GlcNAc modifica- tion. Mass spectrometry-based quantitative interaction proteomics revealed a direct interaction between H2B-S36-O-GlcNAcylated nucleosomes and the TCF20 transcrip- tion factor, suggesting a possible signalling role for the O-GlcNAc modification. Lastly, chapter 5 shows that synthetic histones with reactive side chains can be used to co- valently trap interaction partners in a native context, identifying their binding site by crosslinking mass spectrometry with amino-acid resolution inside the interaction.</p> |
| spellingShingle | Proteomics Biochemistry Bioorganic chemistry Post-translational modification Nadal, S Studying protein interaction and glycosylation with chemical proteomics |
| title | Studying protein interaction and glycosylation with chemical proteomics |
| title_full | Studying protein interaction and glycosylation with chemical proteomics |
| title_fullStr | Studying protein interaction and glycosylation with chemical proteomics |
| title_full_unstemmed | Studying protein interaction and glycosylation with chemical proteomics |
| title_short | Studying protein interaction and glycosylation with chemical proteomics |
| title_sort | studying protein interaction and glycosylation with chemical proteomics |
| topic | Proteomics Biochemistry Bioorganic chemistry Post-translational modification |
| work_keys_str_mv | AT nadals studyingproteininteractionandglycosylationwithchemicalproteomics |