diplomsko delo
Katarina Čubej (Author), Matevž Pompe (Mentor)

Abstract

O-glikozilacija ima zelo pomembno biološko funkcijo in je zato ključna pri raziskovanju bolezni in razvoju bioloških zdravil zanje. Obstajata dva načina za določitev vezavnega mesta O-glikanov na proteine. Prvi je sprostitev O-glikanov z encimskimi in kemičnimi metodami ter obdelava do stabilnejših in označenih derivatov pred analizo. Drugi je razkroj O-glikoproteina, kjer glikani ostanejo pritrjeni na O-glikopeptide, čemur sledi njihovo čiščenje, koncentriranje, separacija in analiza največkrat z različnimi načini fragmentacije v MS. V eksperimentalnem delu sem razvila metodo za separacijo mešanice fluorescenčno označenih O-glikanov s tekočinsko kromatografijo. Metodo na HILIC koloni sem zaradi visokih cen standardov najprej razvila na maltodekstrinih, maltozi in glukozi ter jo nato aplicirala na raztopino pripravljenega standarda. Velik presežek označevalca, ki je motil njihovo separacijo, sem odstranila s SPE ekstrakcijo. V nadaljevanju sem uporabila še kolono z mešanimi režimi, ki je po nekaj modifikacijah omogočila boljše rezultate. Pri validaciji sem preverila linearnost, mejo detekcije in kvantifikacije ter ponovljivost.

Keywords

O-glikani;proteini;vezavno mesto;O-glikolizacija;kromatografija;ekstrakcija na trdno fazo;SPE;validacija;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [K. Čubej]
UDC: 543.544(043.2)
COBISS: 117146115 Link will open in a new window
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Downloads: 22
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Other data

Secondary language: English
Secondary title: Determination of binding site for O-glycan on protein
Secondary abstract: O-glycosylation has important biological function and is therefore key in research of disease and development of biological treatment for it. There are two ways to determine binding site of O-glycans to proteins. The first is release of O-glycans with enzymatic and chemical methods, following treatment to more stable and labeled derivates before analysis. The second is decomposition of O-glycoprotein, where glycans stay intact and binded to O-glycopeptides. After that enrichment, separation and analysis with different types of fragmentation in MS are done. I developed a method for separation of fluorescent-labelled O-glycans with liquid chromatography in experimental part. I first developed method on HILIC column on maltodextrins, maltose and glucose, and then used it on standard solution because of high prices. Excess of label reagent resulted in bad separation, so I removed it with SPE extraction. Later I also used mixed mode chromatography. After few optimization steps this method offered better results. During validation I checked linearity, limit of detection, limit of quantification and repeatability.
Secondary keywords: O-glycans;binding site;chromatography;SPE;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000373
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Kemija
Pages: 44 str.
ID: 15857874