I den här studien har metoder för att separera immunoglobulin (IgG) utvecklats med hjälp av kapillärelektrofores (CE) och Ultra högupplösande vätskekromatografi-Mass spektrometri (UHPLC-MS). Vid analysen av peptider från IgG spjälkat av trypsin (IgG TD) detekterades 26 glykopepeptider med UHPLC-MS genom att använda olika gradienter och mobilfaser. Dessutom analyserades fyra olika koncentrations av IgG TD för att testa detektionsgränsen. Resultatet visade att glykopeptider kunde detekteras i alla fyra koncentrationer men antalet detekterade glykopeptider minskade med minskande koncentration. Den sista delen av UHPLC-MS analysen var en selektivitetsanalys där en blandning av bovint serumalbumin spjälkat av trypsin (BSA TD) och IgG TD analyserades i olika koncentrationer. Selektivitetsanalysen visade att ungefär samma antal IgG glykopeptider detekterades som vid analys av IgG TD. Vid CE-analysen användes tre olika kapillärer, varav två av dem var polyvinylalkohol-coated och olika buffertlösningar (BGE) användes. De slutgiltiga resultaten visade 5 toppar som skulle kunna representera IgG gykopeptider men eftersom ingen MS utfördes kunde inga slutsatser dras. Slutligen, användes Matrix-Assisted Laser desorption/ionisation-time-of-flight (MALDI-TOF) för att analysera proverna innan analys och för att jämföra resultaten med UHPLC-MS. / In this study, methods to separate immunoglobulin G (IgG) glycopeptides were developed using capillary electrophoresis (CE) and ultra-high-performance liquid chromatography-Mass spectrometry (UHPLC-MS). In the UHPLC-MS- analysis, 26 glycopeptides were detected during the analysis of trypsin digested IgG (IgG TD) using different gradients and mobile phases. Moreover, a limit of detection study with four different concentrations (0.01, 0.1, 0.5 and 2mg/mL) was performed. Although the number of detected glycopeptides decreased with decreasing concentration, glycopeptides were detected in the 0.01 mg/mL IgG TD sample. Lastly, a selectivity study was performed, in which two trypsin digested Bovine serum albumin (BSA TD) and IgG TD mixtures with different concentrations were analysed. The results showed that approximately the same number glycopeptides were detected as in the IgG sample suggesting that the BSA sample did not interfere with the detection of glycopeptides. Moreover, the CE-analysis included three different capillaries and several background electrolytes (BGEs). Two of the capillaries were polyvinyl alcohol (PVA) coated. The final electropherogram showed five peaks possibly representing IgG glycopeptides but no MS were performed on the CE part leading to no conclusions for the CE-part. Matrix-Assisted Laser Desorption/Ionisation- time-of flight (MALDI-TOF) was performed throughout the project to control the samples and for comparison to the UHPLC-MS analysis.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-321444 |
| Date | January 2022 |
| Creators | Lövås, Madeleine |
| Publisher | KTH, Kemi |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | TRITA-CBH-GRU ; 2022:301 |
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