Usporedba LC-MS i nano-LC-MS metoda za analizu N-glikozilacije alfa-1 kiselog glikoproteina

master's thesis

• Title (english): Comparison of LC-MS and nano-LC-MS methods for the analysis of N-glycosylation of alpha-1-acid glycoprotein
• Author: Iva Grubeša
• Mentor: Toma Keser (mentor)
• Committee member: Toma Keser (predsjednik povjerenstva)
• Committee member: Najda Rudman (član povjerenstva)
• Committee member: Viktor Pilepić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2024-11-22, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Glikozilacija proteina složena je posttranslacijska modifikacija koja ima mnoge biološke uloge u
organizmu. Abnormalnosti u glikozilaciji primijećene su u nekoliko različitih bolesti te su razlike u
glikozilaciji proteina povezane s osjetljivošću na bolest, tijekom bolesti i odgovorom na terapiju.
Alfa-1 kiseli glikoprotein (AGP) jedan je od glavnih proteina akutne faze kod ljudi. Ima 5 Nglikozilacijskih mjesta, a na ta mjesta kovalentno se vežu različiti tipovi glikana. Nedavno je... More Nglikanski profil AGP-a analiziran nano-LC-MS metodom pri čemu je pokazano da pojedinci s većim
rizikom od dijabetesa imaju povećano grananje AGP N-glikana i nižu sijalinizaciju na nekim
glikozilacijskim mjestima, te bi AGP N-glikanski profil mogao pomoći u razlikovanju pojedinaca
koji su u riziku od razvoja dijabetesa tipa 2.
U ovom radu cilj je bio ispitati može li se glikozilacija AGP-a jednako učinkovito analizirati
korištenjem klasične UHPLC-MS metode koja je jednostavnija, robusnija i lakša za korištenje od
nano-LC-MS-a. AGP je najprije obogaćen iz uzorka precipitacijom „seromukoidne“ frakcije, nakon
čega je uzorak prošao kroz redukciju, alkilaciju i razgradnju enzimom tripsinom. Zatim su
glikopeptidi AGP-a obogaćeni pomoću ekstrakcije na čvrstoj fazi pomoću HILIC zrnaca. Potom su
dobiveni AGP glikopeptidi analizirani pomoću nano-LC-MS-a, te UHPLC-MS-a. Samo su određeni
glikopeptidi prošli parametre kontrole kvalitete (QC), 53 analita u UHPLC-MS-u, a 83 analita u
nano-LC-MS-u. Glikopeptidi koji se preklapaju u obje metode izdvojeni su i normalizirani na
ukupnu integriranu površinu po pojedinom glikozilacijskom mjestu kojem pripadaju. Kako bi se
usporedila ponovljivost između dviju metoda, izračunat je koeficijent varijacije, a kako bi se utvrdila
korelacija, koristio se koeficijent korelacije (r) te je izračunat i koeficijent determinacije (R2).
Pokazano je da obje metode imaju usporedivu ponovljivost, te je vidljiva dobra korelacija u
relativnom intenzitetu većine glikopeptida. U slučaju određenih glikopeptida, pogotovo u onih s
nižim udjelima, ipak postoje razlike u rezultatima. To se može pripisati manjoj osjetljivosti UHPLCMS metode. Unatoč tome, UHPLC-MS je brža, jednostavnija i prikladnija za analizu velikog broja
uzoraka, što je čini idealnom za visoko-propusne analize. Sveukupno, obje metode imaju svoje
mjesto u analizi N-glikozilacije AGP-a, a izbor metode ovisi o specifičnim zahtjevima analize. Less

Abstract (english)

Glycosylation of proteins is a complex post-translational modification with numerous biological
roles in the organism. Abnormalities in glycosylation have been observed in various diseases, and
differences in protein glycosylation are linked to disease susceptibility, disease progression, and
therapeutic response. Alpha-1-acid glycoprotein (AGP) is one of the main acute-phase proteins in
humans. It has 5 N-glycosylation sites, to which various types of glycans are covalently... More attached.
Recently, the N-glycan profile of AGP was analyzed using the nano-LC-MS method, showing that
individuals at higher risk of diabetes have increased branching of AGP N-glycans and reduced
sialylation at certain glycosylation sites, suggesting that the AGP N-glycan profile could help
distinguish individuals at risk of developing type 2 diabetes.
The aim of this study was to determine whether AGP glycosylation can be analyzed just as
effectively using the conventional UHPLC method, which is simpler, more robust, and easier to use
than nano-LC-MS. AGP was first enriched from the sample by precipitating the “seromucoid”
fraction, followed by reduction, alkylation, and digestion with trypsin. AGP glycopeptides were then
enriched using solid-phase extraction with HILIC beads. The AGP glycopeptides were then analyzed
by both nano-LC-MS and UHPLC-MS. Only certain glycopeptides met the quality control (QC)
criteria, with 53 analytes in UHPLC-MS and 83 analytes in nano-LC-MS. Overlapping
glycopeptides from both methods were selected and normalized to the total integrated area of their
respective glycosylation sites. To compare the repeatability of the two methods, the coefficient of
variation was calculated, and the correlation was assessed using the correlation coefficient (r). The
coefficient of determination (R²) was also calculated.
It was shown that both methods have comparable repeatability, with good correlation in the relative
intensity of most glycopeptides. However, for certain glycopeptides, especially those with lower
relative intensity, differences in results were observed. This can be attributed to the lower sensitivity
of the UHPLC-MS method. Despite this, UHPLC-MS is faster, simpler, and more suitable for
analyzing a large number of samples, making it ideal for high-throughput analyses. Overall, both
methods have their place in the analysis of AGP N-glycosylation, and the choice of method depends
on the specific requirements of the analysis. Less

• Keywords: glikozilacija; alfa-1 kiseli glikoprotein; nano-LC-MS; UHPLC-MS
• Keywords (english): glycosylation; alpha-1-acid glycoprotein; nano-LC-MS; UHPLC-MS
• Language: croatian
• URN:NBN: urn:nbn:hr:163:877252
• 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: 2025-02-05 14:32:46