Usporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku

master's thesis

• Title (english): Comparison of microcrystalline cellulose, C18 and HILIC adsorbents for the purpose of purification of human immunoglobulin G glycopeptides for high-throughput glycoproteomics
• Author: Dario Gnjilac
• Mentor: Gordan Lauc (mentor)
• Committee member: Gordan Lauc (predsjednik povjerenstva)
• Committee member: Lidija Bach-Rojecky (član povjerenstva)
• Committee member: Jasna Jablan (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2020-09-10, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Glikozilacija Fc fragmenta humanog imunoglobulina G je vrlo raznolika te varijabilna s obzirom
na različite vanjske i unutarnje utjecaje na organizam. Iz tog razloga se promjene u glikozilaciji i
glikanskim profilima intenzivno istražuju te povezuju s različitim stanjima i bolestima kako bi
poslužile kao potencijalni biomarkeri u dijagnostici ili mete u terapiji. Za osiguravanje ispravnih
zaključaka potrebni su pouzdani i točni podaci dobiveni iz glikobioloških analiza. Budući da takve
analize najčešće podrazumijevaju složene biološke uzorke u kojima su analiti od interesa u vrlo
malim količinama, korak pročišćavanja u procesu analize igra vrlo važnu ulogu.
U ovom su radu uspoređene tri metode pročišćavanja s ciljem dobivanja što konzistentnijih i
točnijih rezultata analize glikopeptida Fc fragmenta humanog imunoglobulina G dobivenih
tripsinizacijom. Uzorak je pročišćavan na tri stacionarne faze: mikrokristaličnoj celulozi te C18 i
HILIC adsorbensu. Nakon pročišćavanja su eluati analizirani LC-MS metodom pri čemu su
dobiveni ekstrahirani ionski kromatogrami s tri grupacije pikova koje odgovaraju podklasama
IgG1, IgG2 i IgG4. Iz svake grupacije su također dobiveni sumirani maseni spektri glikana u
uzorku. Detektirano je dvadeset glikana vezanih za tri različita peptida, dakle sveukupno šezdeset
različitih glikopeptida. Daljnjom analizom podataka programom LaCyTools (v.1.0.1) su dobiveni
podaci o relativnoj zastupljenosti svakog glikopeptida, omjeru signala i šuma te kvaliteti izotopne
raspodjele. Metoda s C18 adsorbensom pokazuje najbolje rezultate u svim promatranim
kategorijama, a kao najvarijabilnijom se pokazala mikrokristalična celuloza. Također je utvrđeno
da je pročišćavanje na C18 adsorbensu financijski najisplativije i vremenski najduže, dok je HILIC
najskuplja opcija, ali nešto kraća po trajanju.

Abstract (english)

The glycosylation of the Fc fragment of human immunoglobulin G is very diverse and variable
depending on different external and internal influences on the organism. For this reason, changes
in glycosylation and glycan profiles are intensively investigated and linked to various conditions
and diseases to serve as potential biomarkers in diagnosis or targets in therapy. Reliable and
accurate data obtained from glycobiological analyzes are required to ensure correct conclusions.
Since such analyzes most often involve complex biological samples in which the analytes of
interest are in very small quantities, the purification step plays a very important role in the analysis
process.
In this paper, three purification methods are compared in order to obtain the most consistent and
accurate results of the analysis of glycopeptides of the Fc fragment of human immunoglobulin G
obtained by trypsinization. The sample was purified on three stationary phases: microcrystalline
cellulose and C18 and HILIC adsorbent. After purification, the eluates were analyzed by the LCMS
method to give extracted ion chromatograms with three groups of peaks corresponding to the
IgG1, IgG2 and IgG4 subclasses. Summarized glycan mass spectra were also obtained from each
group. Twenty glycans bound to three different peptides were detected, ie a total of sixty different
glycopeptides. Further analysis of the data by LaCyTools (v.1.0.1) yielded data on the relative
presence of each glycopeptide, the signal-to-noise ratio, and the quality of the isotope distribution.
The C18 adsorbent method shows the best results in all observed categories, and microcrystalline
cellulose proved to be the most variable. Purification on C18 adsorbent was also found to be the
most cost-effective, while HILIC was the most expensive option, but somewhat shorter in
duration.

• Keywords: pročišćavanje; C18; HILIC; mikrokristalična celuloza; LC-MS; glikopeptidi; ljudski IgG
• Keywords (english): enrichment; C18; HILIC; microcrystalline cellulose; LC-MS; glycopeptides; human IgG
• Language: croatian
• URN:NBN: urn:nbn:hr:163:468557
• 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: 2021-04-30 14:32:10