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The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins
Thesis (MSc)--Stellenbosch University, 2023.
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| Format: | Thesis |
| Language: | en_ZA en_ZA |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613885466411008 |
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| access_status_str | Open Access |
| author | Smith, Logan Jason |
| author2 | Essop, M. Faadiel
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| author_browse | Essop, M. Faadiel
Smith, Logan Jason |
| author_facet | Essop, M. Faadiel
Smith, Logan Jason |
| author_sort | Smith, Logan Jason |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128993 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA en_ZA |
| last_indexed | 2026-06-10T12:43:14.822Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/128993 The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins Smith, Logan Jason Essop, M. Faadiel Joseph, Danzil Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. Post-translational modification Phosphorylation Glycosylation -- Inhibitors Glycoproteins -- Physiological effect Phosphoproteins -- Physiological effect Cardiometabolic diseases -- Effect of stress on Chronic psychosocial stress Protein-protein interactions -- Molecular aspects UCTD Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Introduction: Psychosocial stress has strong links to numerous chronic diseases related to the dysregulated activation of the physiological stress system. This heightens the burden of mortality as there is a robust relationship between chronic psychosocial stress and non- communicable diseases. Hence there is a robust impetus for the identification of novel, circulating biomarkers to earlier detect stress-related chronic diseases. Although protein post-translational modifications such as glycosylation and phosphorylation can act as putative markers of pathophysiology, their relatively low abundance complicates extraction and identification from samples with a high dynamic range. The main aim of this study was therefore to establish two unique enrichment methods for circulatory glycoprotein and phosphoprotein extraction that would then be applied in a preclinical model of chronic psychosocial stress. Methods: Phosphoprotein enrichment was performed using functionalized magnetic particles while glycoprotein enrichment occurred using lectin-bound magnetic particles. Both these methods were tested using a known purified phosphorylated and glycosylated protein and compared to bottom-up proteomics methodology using rat serum. The latter was obtained from a rat model of chronic stress that is well-established in our laboratory (n = 16 controls versus n = 16 stressed rats). These were randomly selected for proteomics analysis to assess the efficiency of retrieval in enriched versus unenriched samples. Fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and proteins visualized using Coomassie and specific fluorescent staining. Here, the relevant Pro-Q stains were employed for the identification of glycosylation and phosphorylation, respectively. The coupling of such enrichment methods to LC – tandem mass spectrometry (MS/MS) was enabled by employing various preparation steps such as deglycosylation and digestion. An exogenous protein was also included as part of the sample preparation to ensure quality control analysis of the LC-MS/MS experiment. Results: SDS-PAGE analyses and staining methods revealed non-specific enrichment with regards to intact protein retrieval. In addition, LC-MS/MS data demonstrated that enrichment using the current set of affinity materials was inadequate for glycopeptide and phosphopeptide retrieval in serum. Conclusion: A lack of enrichment indicates that stringent sample preparation is needed for biological materials with a high dynamic range. This may also be due to the porous nature of both materials employed for phospho- and glycoprotein/peptide enrichment respectively. A combination of enrichment and/or depletion methods may therefore be beneficial for deeper analysis of the blood proteome. These enrichment techniques and the subsequent sample preparation still require further optimization to derive more definitive conclusions in the chronic stress context. AFRIKAANSE OPSOMMING: Inleiding: Psigososiale stres het sterk skakels met talle chroniese siektes wat verband hou met die gedisreguleerde aktivering van die fisiologiese stresstelsel. Dit verhoog die las van sterftes aangesien daar 'n sterk verband tussen chroniese psigososiale stres en nie- oordraagbare siektes is. Daar is dus 'n sterk motivering vir die identifisering van nuwe, sirkulerende biomerkers om stresverwante chroniese siektes vroeër op te spoor. Alhoewel proteïen-post-translasionele modifikasies soos glikosilasie en fosforilering as herkende merkers van patofisiologie kan dien, bemoeilik hul relatief lae hoeveelheid ekstraksie en identifikasie vanuit monsters met 'n hoë dinamiese omvang. Die hoofdoel van hierdie studie was dus om twee unieke verrykingsmetodes vir sirkulatoriese glikoproteïen- en fosfoproteïenontginning daar te stel, wat dan in 'n prekliniese model van chroniese psigososiale stres toegepas sou word. Metodes: Fosfoproteïenverryking is uitgevoer met behulp van gefunksioneerde magnetiese partikels, terwyl glikoproteïenverryking plaasgevind het met behulp van lektiengebonde magnetiese partikels. Albei hierdie metodes is getoets met behulp van 'n bekende gesuiwerde gefosforileerde en glikosileerde proteïen en in vergelyking met onder-na-bo proteomiese-metodologie met behulp van rotserum. Laasgenoemde is verkry uit 'n rotmodel van chroniese stres wat goed gevestig is in ons laboratorium (n = 16 kontroles teenoor n = 16 gestresde rotte). Dit is lukraak gekies vir proteomiese-analise om die doeltreffendheid van herwinning in verrykte teenoor onverrykte monsters te bepaal. Fraksies is ontleed deur natriumdodesielsulfaat poliakrylamiedgelelektroforese (SDS-PAGE) en proteïene gevisualiseer met behulp van Coomassie en spesifieke fluoresserende kleuring. Hier is die relevante Pro-Q-kleurstowwe gebruik vir die identifisering van onderskeidelik glikosilasie en fosforilering. Die koppeling van sulke verrykingsmetodes aan vloeistofchromatografie (VC) - tandemmassaspektrometrie (MS/MS) is moontlik gemaak deur gebruik te maak van verskillende voorbereidingsstappe soos deglikosilasie en vertering. ‘n Eksogene proteïen is ook ingesluit as deel van die monstervoorbereiding om kwaliteitsbeheeranalise van die VC-MS/MS-eksperiment te verseker. Resultate: SDS-PAGE-ontledings en kleuringsmetodes het nie-spesifieke verryking met betrekking tot ongeskonde proteïenherwinning aan die lig gebring. Daarbenewens het VC- MS/MS-data getoon dat verryking met behulp van die huidige stel affiniteitsmateriaal onvoldoende was vir glikopeptied- en fosfopeptied-herwinning in serum. Gevolgtrekking: 'n Gebrek aan verryking dui daarop dat streng monstervoorbereiding nodig is vir biologiese materiale met 'n hoë dinamiese omvang. Dit kan ook te wyte wees aan die poreuse aard van beide materiale wat onderskeidelik vir fosfoproteïen- en glikoproteïenverryking gebruik word. 'n Kombinasie van verrykings- en/of uitputtingsmetodes kan dus voordelig wees vir dieper ontleding van die bloedproteoom. Hierdie verrykingstegnieke en die daaropvolgende steekproefvoorbereiding vereis steeds verdere optimalisering om meer definitiewe gevolgtrekkings in die chroniese streskonteks af te lei. Masters 2023-11-27T10:42:34Z 2024-01-08T18:30:41Z 2023-11-27T10:42:34Z 2024-01-08T18:30:41Z 2023-12 Thesis https://scholar.sun.ac.za/handle/10019.1/128993 en_ZA en_ZA Stellenbosch University xix, 229 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Post-translational modification Phosphorylation Glycosylation -- Inhibitors Glycoproteins -- Physiological effect Phosphoproteins -- Physiological effect Cardiometabolic diseases -- Effect of stress on Chronic psychosocial stress Protein-protein interactions -- Molecular aspects UCTD Smith, Logan Jason The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins |
| title | The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins |
| title_full | The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins |
| title_fullStr | The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins |
| title_full_unstemmed | The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins |
| title_short | The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins |
| title_sort | detection of stress related diseases establishment of two unique methods to discover circulatory phospho and glycoproteins |
| topic | Post-translational modification Phosphorylation Glycosylation -- Inhibitors Glycoproteins -- Physiological effect Phosphoproteins -- Physiological effect Cardiometabolic diseases -- Effect of stress on Chronic psychosocial stress Protein-protein interactions -- Molecular aspects UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/128993 |
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