Optimizacija visokoprotočne metode za analizu N-glikozilacije alfa-1 kiselog glikoproteina

master's thesis

• Title (english): Optimization of high-throughput method for N-glycosylation analysis of human alpha-1 acid glycoprotein
• Author: Maja Ukalović
• Mentor: Toma Keser (mentor)
• Committee member: Toma Keser (predsjednik povjerenstva)
• Committee member: Olga Gornik Kljaić (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: 2021-09-16, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Alfa-1 kiseli glikoprotein (AGP) izrazito je glikoziliran serumski protein akutne faze koji sadrži pet vezanih N-glikana kompleksnog tipa na polipeptidnoj okosnici. Promjene u glikozilaciji AGP-a prisutne u raznim fiziološkim i patofiziološkim stanjima. Nedavno je razvijena nova ekonomski isplativa visokoprotočna metoda koja omogućuje precizno i sveobuhvatno određivanje N-glikanskog profila AGP-a iz malog volumena krvnog seruma ili plazme. Navedenom metodom pokazalo se da je N-glikanski profil stabilan kod zdravih pojedinaca, a promjene u njegovoj N-glikozilaciji bi mogle poslužiti kao novi biomarker u prepoznavanju osoba s povećanim rizikom za razvoj diabetes mellitusa tipa II.
AGP se obogaćuje iz uzorka precipitacijom seromukoidne frakcije nakon koje slijedi redukcija, alkilacija i tripsinizacija te daljnje obogaćivanje glikopeptida pomoću ekstrakcije na čvrstoj fazi (HILIC-SPE). Dobiveni glikopeptidi analiziraju se obrnuto faznom kromatografijom spregnutom s masenim spektrometrom čime se dobiva detaljan uvid u glikanski profil svakog N-glikozilacijskog mjesta AGP-a. Prilikom provođenja metode na velikom broju uzoraka, kod nekih je primijećen smanjen intenzitet signala glikozilacijskog mjesta III.
Kako bi se utvrdio uzrok navedenog odstupanja ispitan je utjecaj koncentracije tripsina i udjela ACN-a u mobilnoj fazi HILIC-SPE na intenzitet glikozilacijskog mjesta III. Utjecaj tripsina je ispitan zbog otpornosti AGP-a na tripsinizaciju, dok je povećanje udjela ACN-a u mobilnoj fazi razmotreno zbog velikog udjela hidrofobnog peptidnog djela koji bi mogao smanjiti afinitet prema polarnoj stacionarnoj fazi prilikom obogaćivanja ekstrakcijom na čvrstoj fazi.
Promjena koncentracije tripsina nije utjecala na intenzitet signala, dok je povećanje udjela ACN-a u koraku obogaćivanja pojačalo intenzitet signala glikozilacijskog mjesta od interesa. Ove promjene u protokolu (osim prilikom korištenja pet puta veće koncentracije tripsina od standardne) smanjile su preciznost u odnosu na standardnu metodu, što u konačnici nije pogodno za optimizaciju metode. U budućem radu potrebno je ispitati druge stacionarne faze koje bi bile prikladne za korištenje prilikom HILIC-SPE ili neke druge metode obogaćivanja koje bi osim povećanje intenziteta signala glikozilacijskog mjesta III zadovoljile uvjete preciznosti metode u odnosu na standardni postupak.

Abstract (english)

Alpha-1-acid glycoprotein (AGP) is an acute-phase plasma protein with high carbohydrate content attached in the form of five N-linked complex glycans. Altered glycosilation of AGP occurs in many physiological and pathophysiological conditions. Recently, a cost-effective method for a high-throughput detailed AGP N-glycosylation profiling was developed, which provides site-specific glycosylation information from a small volume of blood serum or plasma. Using the method, it was demonstrated that N-glycan profile of AGP is stable in a healthy individual and that changes in N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.
AGP is enriched from blood plasma by acid precipitation of seromucoid fraction, after which it undergoes reduction, alkylation and trypsinization to be further enriched using solid-phase extraction (HILIC-SPE). Glycopeptides obtained by this process are then analysed by reversed-phase liquid chromatography paired with electrospray ionization-MS. During the implementation of the method on large cohorts, reduced intensity of glycosylation site III signal was observed in a certain number of measurements.
To determine the cause of irregularity, influence of trypsin concentration and increased concentration of acetonitrile in HILIC mobile phase on intensity of glycosylation site III signal was evaluated. The effect of trypsin was evaluated due to AGP being resistant to conventional tryptic digestion, whereas a higher concentration of ACN was investigated because of an extensive amount of hydrophobic peptide moiety of glycosylation site III which could lower the affinity towards the stationary phase in HILIC-SPE enrichment
The change in trypsin concentration didn't affect the intensity of the signal, whereas a higher concentration of ACN has enhanced the signal intensity of the glycosylation site of interest. These changes in the protocol (except for the trypsin concentration that was five times higher than the standard) have resulted in decreased precision compared to the standard method. This decrease is not suitable for the method optimisation. In further research, other stationary phases that could be suitable for HILIC-SPE enrichment or different enrichment methods should be tested in order to find a method that would also meet the precision criteria while increasing the signal intensity of the glycosylation site III.

• Keywords: alfa-1 kiseli glikoprotein; N-glikozilacija; HILIC-SPE; LC-MS; optimizacija
• Keywords (english): alpha-1 acid glycoprotein; N-glycosilation; HILIC-SPE; LC-MS; optimization
• Language: croatian
• URN:NBN: urn:nbn:hr:163:385345
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije i laboratorijske medicine (magistar/magistra medicinske biokemije i laboratorijske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-09-16
• Created on: 2022-03-28 09:07:47