Glycan mapping method based on UPLC
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
To get the possibility to identify structures of different O-glycans, a glycan mapping method has been created and developed. Using non-reductive β-elimination, which is a chemical release, the O-linked carbohydrates were released from glycoproteins.
Pichia pastoris is a methylotrophic yeast, which is used as a platform for production of therapeutic glycoproteins. Recopharma produces glycoproteins from P.pastoris. These glycoproteins are called P-PM and were used for studies in this project.
Further, the released carbohydrates were labeled with a chromophore to increase the detection capacity in an ultra performance liquid chromatography, UPLC. To succeed with the labeling the glycan sample was purified from detergents, remaining proteins and salt, that can disturb the labeling reaction. To increase the purification and further succeed with the labeling reaction a solid phase extraction (SPE) process, was used on the carbohydrates before the glycan labeling reaction. To see how well the SPE-cartridge could purify the glycan sample mass spectrometry data were compared for a glycan sample that was treated with a SPE-cartridge with one that was not.
The MS analyses showed that the SPE-cartridge managed to clean the glycan sample. So, by using a SPE-cartridge, the labeling reaction and the detection in UPLC would succeed.
Two different columns were available for the UPLC-equipment. To get an idea of which of these columns that were suitable for this purpose, the columns were compared. The chromatograms for runs with respective column were fairly different and it was decided to continue the optimization of the glycan mapping method, with the column that gave the best separation.
Place, publisher, year, edition, pages
2011. , 50 p.
glycans, glycoproteins, mapping, ultra performance liquid chromatography, UPLC
IdentifiersURN: urn:nbn:se:kth:diva-33658OAI: oai:DiVA.org:kth-33658DiVA: diva2:416899
Subject / course
Master of Science in Engineering
UppsokPhysics, Chemistry, Mathematics
Gustafsson, AnkiJohansson, Tomas