Razvoj visokoprotočne metode za analizu N-glikozilacije fibrinogena

master's thesis

• Title (english): Development of a high-throughput method for the N-glycosylation analysis of fibrinogen
• Author: Franka Knežević
• Mentor: Dinko Šoić (mentor)
• Committee member: Dinko Šoić (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: 2025-04-03, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Glikozilacija je jedna od najvažnijih posttranslacijskih modifikacija koja značajno utječe na biološke funkcije i strukturu proteina. Fibrinogen, ključni protein u procesu zgrušavanja krvi, podliježe N-glikozilaciji na glikozilacijskim mjestima Asn394 na β-lancu i Asn78 na γ-lancu. Analiza N-glikozilacije fibrinogena mogla bi pružiti uvid u potencijalne glikozilacijske promjene ovog proteina u različitim patološkim stanjima poput KOPB-a i kardiovaskularnih bolesti. Cilj ovog istraživanja bio je stoga razviti visokoprotočnu metodu za analizu N-glikozilacije fibrinogena iz uzorka krvne plazme. Obogaćivanje fibrinogena provedeno je precipitacijom etanolom, a glikozilacija je analizirana na razini glikopeptida.
Ispitano je šest različitih eksperimentalnih uvjeta za pripremu i analizu glikopeptida fibrinogena. Za svaki uvjet, MS1 analizom uspoređeni su apsolutni signali glikopeptida, pri čemu je eksperimentalni uvjet s 16% etanola u odnosu na volumen plazme pokazao najvišu osjetljivost i reproducibilnost analize. Za potvrdu glikoformi korištena je MS/MS analiza, pri čemu je fragmentacija najdominantnije glikoforme (A2G2S1) na oba glikozilacijska mjesta pokazala očuvanje peptidne sekvence uz karakterističan raspad glikanske strukture. Na temelju poznatih podataka iz literature o glikanskim strukturama fibrinogena te retencijskih vremena njihovih glikopeptida, identificirane su i potvrđene dvije najzastupljenije glikoforme (A2G2S1 i A2G2S2) na oba glikozilacijska mjesta, kao i dodatne glikoforme: A2G2, FA2G2S1 i FA2G2S2 na β-lancu te FA2G2S1 i FA2G2S2 na γ-lancu.
Ovo istraživanje uspješno je razvilo visokoprotočnu metodu za N-glikoprofiliranje fibrinogena iz krvne plazme te omogućilo detaljno određivanje njegovih glikanskih struktura i glikozilacijskog mjesta s kojeg potječu. Dobiveni rezultati pružaju temelj za daljnja istraživanja u području glikoproteomike te potencijalnu primjenu u identifikaciji biomarkera i proučavanju uloge glikozilacije fibrinogena u različitim bolestima.

Abstract (english)

Glycosylation is one of the most important post-translational modifications that significantly affects the biological functions and structure of proteins. Fibrinogen, a key protein in the blood coagulation process, undergoes N-glycosylation at glycosylation sites Asn394 on the β-chain and Asn78 on the γ-chain. Analyzing fibrinogen N-glycosylation could provide insight into potential glycosylation changes in various pathological conditions such as COPD and cardiovascular diseases. The aim of this research was to develop a high-throughput method for analyzing fibrinogen N-glycosylation from a blood plasma sample. Fibrinogen enrichment was performed by ethanol precipitation, and glycosylation was analyzed at the glycopeptide level.
Six different experimental conditions for fibrinogen glycopeptide preparation and analysis were tested. For each condition, absolute glycopeptide signals were compared by MS1 analysis, with the experimental condition with 16% ethanol relative to plasma volume showing the highest sensitivity and reproducibility of the analysis. MS/MS analysis was used to confirm the glycoforms, with the fragmentation of the most dominant glycoform (A2G2S1) at both glycosylation sites showing peptide sequence preservation along with the characteristic fragmentation of the glycan structure. Based on known literature data on fibrinogen glycan structures and the retention times of their glycopeptides, two most abundant glycoforms (A2G2S1 and A2G2S2) were identified and confirmed at both glycosylation sites, as well as additional glycoforms: A2G2, FA2G2S1 and FA2G2S2 on the β-chain, and FA2G2S1 and FA2G2S2 on the γ-chain.
This study successfully developed a high-throughput method for N-glycoprofiling of fibrinogen from blood plasma, enabling detailed characterization of its glycan structures and their glycosylation sites of origin. The obtained results provide a foundation for further research in the field of glycoproteomics and potential application in biomarker identification and studying the role of fibrinogen glycosylation in various diseases.

• Keywords: fibrinogen; N-glikani; N-glikozilacija; glikoprotein; masena spektrometrija
• Keywords (english): fibrinogen; N-glycans; N-glycosylation; glycoprotein; mass spectrometry
• Language: croatian
• URN:NBN: urn:nbn:hr:163:640050
• Study programme: Title: Pharmacy - integrated undergraduate and graduate - university study; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: sveučilišni magistar farmacije (sveučilišni magistar farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2025-04-16 08:48:59