Utjecaj smrzavanja i odmrzavanja na N-glikom imunoglobulina G i krvne plazme

master's thesis

• Title (english): Effects of freezing and thawing on immunoglobulin G and human plasma N-glycome
• Author: Zlatko Ficović
• Mentor: Gordan Lauc (mentor)
• Committee member: Gordan Lauc
• Committee member: Olga Gornik
• Committee member: Suzana Inić
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2015-07-08, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE

Abstract

Glikozilacija je široko rasprostranjena posttranslacijska modifikacija koja omogućuje značajan broj strukturnih promjena bioloških molekula. Istraživanja su pokazala da se glikozilacija in vivo može promijeniti pod utjecajem vanjskih čimbenika ili u patološkim stanjima. No, do promjene N-glikoma može doći i in vitro pod utjecajem čimbenika poput pH ili temperature. To je u analitici glikana vrlo značajno jer se biološki uzorci čuvaju pri nižim temperaturama, a često se i smrzavaju. U ovom radu analiziran je N-glikom proteina ljudske krvne plazme i imunoglobulina G nakon više ciklusa smrzavanja (-20 oC i -80 oC) i odmrzavanja (+37 oC). Nadalje, analizirani su i čisti fragmenti IgG Fc G0F i G2F. IgG je iz krvne plazme izoliran pomoću monolita s proteinom G. N-glikani su oslobođeni s proteina pomoću PNGaze F te su potom obilježeni 2-aminobenzamidom, koji je fluorescentna boja. Kromatogrami dobiveni nakon analize na UPLC-HILIC pokazuju da s brojem ciklusa smrzavanja i odmrzavanja raste varijabilnost glikozilacije ukupnih proteina plazme, imunoglobulina G, kao i fragmenata IgG Fc G0F i G2F. Nema značajnih razlika ako se međusobno uspoređuju rezultati dobiveni na -20 oC i -80 oC. Kod uzoraka plazme primijećeno
je razdvajanje slojeva nakon desetak ciklusa smrzavanja. Donji sloj sadrži većinu proteina iz uzorka, a profil glikana je različit u slojevima. Udio glikana G1F (oba izomera) u uzorcima se povećava, dok se udio G2F smanjuje. No, udio G0F se ne mijenja. Postoji mogućnost da G2F gubi jednu molekulu galaktoze te prelazi u G1F. Drugu molekulu galaktoze ne gubi, a mogući razlog za to je nepovoljna konformacijska promjena proteina nakon gubitka prve molekule galaktoze. Potrebno je napraviti daljnja istraživanja kako bi se došlo do konačnog zaključka o mehanizmu deglikozilacije uslijed smrzavanja uzorka.

Abstract (english)

Glycosylation is an abundant posttranslational modification that allows creating a wide range of structural changes in biomolecules. Studies have shown that glycosylation in vivo can be modified under environmental influence or pathological conditions. Also, N-glycome changes can happen in vitro under influence of factors, such as pH or temperature. That fact is very significant in glycan analysis because biological samples are stored on lower temperatures, and are often frozen. In this research N-glycome of human plasma proteins and immunoglobulin G was analyzed, after several freezing (at -20 oC and -80 oC) – thawing (at +37 oC) cycles. Furthermore, glycans of fragments IgG Fc G0F and G2F were analyzed. IgG was isolated from human plasma using protein G monolithic plate. N-glycans were released from proteins using PNGase F and consequently labeled with 2-aminobenzamide, which is a fluorescent dye. Chromatograms obtained after the UPLC-HILIC analysis show that glycosylation variability increases with the number of freezing-thawing cycles. That was noticed in all three types of samples: human plasma proteins, IgG pool and IgG Fc G0F+G2F. There is no significant difference if results obtained at -20 oC and -80 oC are compared. G1F fraction (both isomers) in IgG Fc G0F+G2F samples increases, while G2F fraction decreases. But, G0F fraction does not change. Further research is needed to reach a final conclusion regarding mechanism of protein deglycosylation due to freezing of samples.

• Keywords: glikozilacija; N-glikom; imunoglobulin G; plazma; smrzavanje
• Keywords (english): glycosylation; N-glycome; immunoglobulin G; human plasma; freezing
• Language: croatian
• URN:NBN: urn:nbn:hr:163:309380
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2016-01-19 15:54:48