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Effect of elution solution on the enrichment and purification of glycopeptides using SPE-HILIC
Chobotová, Michaela ; Kozlík, Petr (advisor) ; Křížek, Tomáš (referee)
Changes in protein glycosylation are related to various diseases such as cancer or Alzheimer's disease. Unfortunately, the analysis of glycoproteins remains a difficult task due to the low proportion of glycopeptides compared to peptides obtained after glycoprotein cleavage. As a result, it is almost impossible to analyze glycopeptides without specific enrichment steps. A suitable method for the enrichment of glycopeptides is solid phase extraction (SPE) using the principle of hydrophilic interaction liquid chromatography (HILIC). Since the choice of solvent significantly affects the enrichment of glycopeptides, the aim of this thesis was to compare the effect of different solvents on the efficiency of glycopeptide extraction using SPE-HILIC. The extraction was carried out on a silica column modified with aminopropyl groups. Model analytes were glycopeptides obtained by tryptic digestion of human IgG. During the comparsion of acetonitrile, methanol and isopropanol, acetonitrile was chosen as a suitable elution solvent. During the conditioning and washing step, the content of acetonitrile was changed (65%, 75% and 85%), whereby the elution of glycopeptides is accelerated as the amount of acetonitrile decreases. In the elution step, the effectiveness of different acetonitrile contents (5%, 10% and...
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