Usporedba obilježavanja N-glikana 2-aminobenzamidom i prokainamidom u analizi tekućinskom kromatografijom ultravisoke djelotvornosti temeljenoj na hidrofilnim interakcijama

master's thesis

• Title (english): Comparison of procainamide and 2-aminobenzamide labeling for analysis of N-glycans using HILIC-UPLC
• Author: Isabella Zolić
• Mentor: Olga Gornik (mentor)
• Committee member: Olga Gornik (predsjednik povjerenstva)
• Committee member: Toma Keser (član povjerenstva)
• Committee member: Ana Mornar Turk (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2017-05-25, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Ovaj se rad bavi validacijom HILIC-UPLC metode u svrhu analize N-glikana. Glikozilacija je modifikacija proteina od esencijalne važnosti za njihovu strukturu i funkciju. Zahvaljujući ovom saznanju te shvaćanjem da glikozilacija ima veoma važnu ulogu u fiziologiji i patofiziologiji, analiza glikana se sve više uključuje u biološka istraživanja, kliničke analize te u farmaceutskoj proizvodnji. Brojne analitičke metode su se razvile u svrhu ispitivanja glikana. Jedna od metoda jest HILIC-UPLC analiza izoliranih i fluorescentno obilježenih glikana, koja omogućuje relativnu kvantifikaciju različitih glikana na temelju intenziteta fluorescencije. U tu se svrhu često koristi 2-aminobenzamid. Velika primjenjivost ove tehnike u analizi glikana sugerira validaciju metode u svrhu poboljšanja analize. Stoga su u ovom istraživanju ispitana svojstva prokainamida kao potencijalnog fluorescentnog obilježivača.
Rezultati ovog rada pokazuju da se fluorofor prokainamid moţe koristiti u analizi glikana. Obilježavanjem N-glikana s 2-aminobenzamidom i prokainamidom dobivena su dva usporediva glikanska profila s 24 vrška što ukazuje na dosljednost i primjenjivost prokainamida. Prednost prokainamida u odnosu na 2-aminobenzamid je veća osjetljivost i niži limit kvantifikacije koje će nam uz gotovo jednaku preciznost omogućiti relativnu kvantifikaciju glikana nižih masa od onih obilježenih 2-aminobenzamidom.

Abstract (english)

This Master thesis deals with the validation of the HILIC-UPLC method of N-glycan analysis. Glycosylation is the process of protein modification essential to their structure and function. Due to advances in the research of glycans, glycosylation has been recognized as an important part of physiology and pathophysiology. Glycan analysis is becoming more important in biological research, clinical analysis and pharmacy. Numerous analytical methods have been developed for glycan research. One such method is the HILIC-UPLC analysis of isolated and fluorescently marked glycans, which makes relative quantification of different glycans based on fluorescence intensity possible. 2-aminobenzamide is often used label glycans. The broad use of this technique in glycan analysis calls for validation of the method, which has the potential to improve analyses. Therefore, this paper researches the qualities of procainamide as a potential fluorescent label.
The results show that fluorophore procainamide can be used in glycan anaylsis. Labelling N-glycans with 2-aminobenzamide and procainamide resulted in two comparable glycan profiles with 24 peaks, which proves the consistency and applicability of procainamide. The advantage of procainamide, when compared to 2-aminobenzamide, lies in its greater sensitivity and lower quantification limits. These qualities allow for precision almost equal to 2-aminobenzamide in the relative quantification of lower glycan concentrations.

• Keywords: N-glikani; imunoglobulin G; HILIC-UPLC; 2-aminobenzamid; prokainamid
• Keywords (english): N-glycans; Immunoglobulin G; Prokainamide; 2-aminobenzamide; HILIC-UPLC
• Language: croatian
• URN:NBN: urn:nbn:hr:163:512617
• 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: Open access
• Created on: 2018-11-28 15:40:06