Usporedba efikasnosti obilježavanja N-glikana imunoglobulina G 2-aminobenzamidom, prokainamidom i RapiFluor-MS-om

Glycosylation is a complex cotranslational and posttranslational reaction of enzyme-mediated modification, in which the sugar structures covalently bind to proteins and lipids, and glycans, as a structural part of glycoproteins, are of utmost importance for their function and proper structure and are being investigated more and more today. One of the most frequently used glycan analytical techniques is ultra performance liquid chromatography based on hydrophilic reactions (HILIC-UPLC) coupled with mass spectrometry. In order for the glycans to be detected, their analysis requires fluorescence labeling. This Master thesis compares the efficacy of labeling immunoglobulin G's N-glycans with three fluorescent dyes- 2-aminobenzamide, procainamide and RapiFluor-MS. It examines how much impact on the results of the analysis has the success of binding fluorescent dye to N-glycans, irrespective of its ability to ionize. Comparing the absolute intensity ratios of unlabeled glycans in samples obtained by mass spectrometry (ESI-QToF-MS), it is concluded that the efficacy of fluorescent dye binding for N-glycans is practically identical, since all ratios are approximately equal to 1, meaning that there is no significant influence of binding efficacy on the results of analysis regarding different peak intensities. Also, the range of the amount of starting IgG in the sample, in which the N-glycan labeling is the most efficient, is 25-50 μg for 2-aminobenzamide, in procainamide is 5-50 μg and for RapiFluor-MS is ranging from 2 to 15 g. These results confirm that, considering sensitivity, 2-aminobenzamide is the worst and RapiFluor-MS is the best choice for labeling N-glycans in their mass spectrometric analysis.