Development of a LC-MS/MS method for the detection of snake venom toxins in human plasma

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dc.contributor.advisorKellermann, Tracyen_ZA
dc.contributor.authorLermer, Anneen_ZA
dc.contributor.otherFaculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.en_ZA
dc.date.accessioned2022-11-08T08:29:07Zen_ZA
dc.date.accessioned2023-01-16T12:42:24Zen_ZA
dc.date.available2022-11-08T08:29:07Zen_ZA
dc.date.available2023-01-16T12:42:24Zen_ZA
dc.date.issued2022-12en_ZA
dc.descriptionThesis (MSc) -- Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH ABSTRACT: Background: Snakebite envenomation in sub-Saharan Africa significantly threatens human health. Species that was responsible for the envenomation are rarely positively identified. There are no rapid methods/diagnostics available for conclusive identification of the organism/species responsible. Venomous species can be categorised based on the composition and primary effect caused by their venom components as neurotoxic, cytotoxic and/or hemotoxic. Aim: To develop an LC-MS/MS method for the species-specific detection of snake venom toxins from human plasma. Methods: An epidemiological study on the incidence of snakebite in South Africa as reported to Poisons Information Helpline of Western Cape (PIHWC) was performed to provide rationale for the analytical study. Analytical methodology includes identification of species-specific venom toxins by high resolution (HR) LC-MS/MS, fractionation of Naja nivea venom by size exclusion chromatography and compositional analysis of the fractions by HR-LC-MS/MS. Venom fractions were screened for toxicity using a zebrafish model, utilising DanioVision for phenotypic screening and behavioural tracking of the larvae. Thereafter, a triple quadrupole LC-MS/MS method was developed containing species-specific toxins. Results: Analysis of PIHWC call data revealed that in 43.73% of the recorded snakebite cases the causative snake species were unidentified. The snake species Naja nivea, Bitis arietans and Bitis atropos species were identified as most medically significant in South Africa and consequently included in the study. Venom from N. nivea was chosen as the species for fractionation after common peptides from inter-region venom were identified as unique to the species. Behavioural changes in larvae were observed after exposure to sub-lethal concentrations of N. nivea venom fractions. Significant (p<0.0001) changes in larval behaviour were observed in two treatment groups compared to the control. Using transitions that was generated during HR-LC-MS/MS analysis, FASTA files were generated and converted into MRM’s onto the triple quadrupole LC-MS. Toxins were positively identified from human plasma by LC-MS/MS. Discussion: This study identified the species predominantly responsible for snakebites from PIHWC call data, and highlighted a need for a diagnostic to identify the species responsible for envenomation. Analysis of the venom proteomes by HR-LC-MS/MS revealed similarities and differences in the venom profiles of Naja nivea, Bitis arietans and Bitis atropos species. In-solution, HILIC assisted tryptic digestion produced the identification of more proteins from the N. nivea crude venom compared to in-gel digestion and while fractionation by size exclusion chromatography prior to MS analysis increased the detection of low molecular weight toxins. It was also shown that using a combination of conventional HR-MS (with database/library searches) and triple quadrupole MS, a method could be created that identified species specific venom peptides from human plasma to aid diagnosis. Conclusion: This is a proof of concept for future work that will include the development of a lateral flow assay to detect venom from envenomed plasma that is cost-effective to produce, aids in defining diagnosis and importantly serves victims of snakebite envenomation in rural communities.en_ZA
dc.description.abstractAFRIKAANS OPSOMMING: Agtergrond: Slangbytvergiftiging in Afrika suid van die Sahara hou ʼn beduidende bedreiging vir menslike gesondheid in. Die spesie wat vir die vergiftiging verantwoordelik is, word selde positief uitgeken. Daar is geen vinnige metodes/diagnostiek beskikbaar vir voldoende uitkenning van die verantwoordelike organisme/spesie nie. Giftige spesies kan op grond van die samestelling en primêre effek van hulle gif as neurotoksies, sitotoksies en/of hemotoksies gekategoriseer word. Doel: Om ʼn vloeistofchromatografie-massaspektografie- (LC-MS/MS) metode van spesie-spesifieke opsporing van slanggiftoksiene uit menslike plasma te ontwikkel. Metodes: ʼn Epidemiologiese studie oor die voorkoms van slangbyt in Suid-Afrika, soos by die Wes-Kaapse Gifinligtingshulplyn (PIHWC) aangemeld, is gedoen om die beweegrede vir die analitiese studie te bied. Analitiese metodologie sluit in die identifisering van spesie-spesifieke giftoksiene deur hoëresolusie- (HR-) LC-MS/MS, fraksionering van die gif van Naja nivea deur grootte-uitsluitingschromatografie en samestellingsontleding van die fraksies deur HR-LC-MS/MS. Giffraksies is vir giftigheid getoets deur van ʼn sebravismodel gebruik te maak, met behulp van DanioVision vir fenotipiese sifting en gedragstredhou van die larwes. Daarna is ʼn drievoudige vierpool-LC-MS/MS-metode ontwikkel, wat spesie-spesifieke toksiene bevat. Uitslae: Ontleding van die oproepdata van die PIHWC getoon dat in 43.73% van die aangemelde slangbytgevalle die spesie van die betrokke slang nie uitgeken is nie. Die slangspesies Naja nivea, Bitis arietans en Bitis atropos is uitgeken as die medies mees beduidende in Suid-Afrika en is gevolglik by die studie ingesluit. Gif uit N. nivea is gekies as die spesie vir fraksionering, nadat gedeelde peptiede uit tussenstreekse gif as uniek tot die spesies geïdentifiseer is. Gedragsverandering is in larwes waargeneem ná blootstelling aan subdodelike konsentrasies van giffraksies van N. nivea. Beduidende (p<0.0001) veranderinge in larwegedrag is waargeneem in die twee behandelingsgroepe, vergeleke met die kontrolegroep. Met behulp van die m/z-oorgange wat tydens die HR-LC-MS/MS-ontleding gegenereer is, is FASTA-lêers gegenereer en omgeskakel na meervoudige reaksiemonitering (MRM) op die drievoudige vierpool-LC-MS. Toksiene is positief met behulp van LC-MS/MS uit menslike plasma geïdentifiseer. Bespreking: Die studie identifiseer die drie medies-beduidende spesies wat hoofsaaklik vir slangbyte verantwoordelik is en hoe dikwels die oorsaakorganisme volgens die PIHWS-data onbekend is. Dit lig die behoefte aan diagnostiek vir die uitkenning van die spesie wat vir die vergiftiging verantwoordelik is, uit. Ontleding van die gifproteome deur HR-LC-MS/MS onthul ooreenkomste en verskille in die gifprofiele van Naja nivea, Bitis arietans en Bitis atropos. In-oplossing, HILIC-gesteunde (hidrofiliese interaksie vloeistofchromatografie) triptiese vertering identifiseer nog proteïene uit onverwerkte N.nivea-gif vergeleke met in-jel-vertering. Daar word ook getoon dat, met die gebruik van ʼn kombinasie van konvensionele HR-MS (met databasis-/biblioteeksoektogte) en drievoudige vierpool-MS, ʼn metode geskep kan word wat spesie-spesifieke gifpeptiede uit menslike plasma kan identifiseer om met diagnose te help. Gevolgtrekking: Dit is ʼn bewys van die konsep vir toekomstige werk, insluitend die ontwikkeling van ʼn laterale vloeitoets vir die opspoor van gif uit vergiftigde plasma, wat kostedoeltreffend is om te vervaardig, help met die definiëring van diagnose en die slagoffers van slangbytvergiftiging in landelike gemeenskappe kan dien.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxviii, 177 pages : illustrationsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/125944en_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectPoisonous snakes – Venom – Toxicology – South Africaen_ZA
dc.subjectSnakebites -- Treatment – South Africa – South Africaen_ZA
dc.subjectPoisonous snakes – Venom -- Therapeutic use – South Africaen_ZA
dc.subjectCytotoxicity – South Africaen_ZA
dc.subjectVenom – Toxicology – South Africaen_ZA
dc.subjectUCTDen_ZA
dc.titleDevelopment of a LC-MS/MS method for the detection of snake venom toxins in human plasmaen_ZA
dc.typeThesisen_ZA
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