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master's thesis
Usporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku
Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry, 2020. urn:nbn:hr:163:468557
Gnjilac, Dario
University of Zagreb
Faculty of Pharmacy and Biochemistry
Department of biochemistry and molecular biology

Institutional repository: Repository of Faculty of Pharmacy and Biochemistry University of Zagreb

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Gnjilac, D. (2020). Usporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku (Master's thesis). Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry. Retrieved from https://urn.nsk.hr/urn:nbn:hr:163:468557

Gnjilac, Dario. "Usporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku." Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, 2020. https://urn.nsk.hr/urn:nbn:hr:163:468557

Gnjilac, Dario. "Usporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku." Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, 2020. https://urn.nsk.hr/urn:nbn:hr:163:468557

Gnjilac, D. (2020). 'Usporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku', Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, accessed 30 December 2024, https://urn.nsk.hr/urn:nbn:hr:163:468557

Gnjilac D. Usporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku [Master's thesis]. Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry; 2020 [cited 2024 December 30] Available at: https://urn.nsk.hr/urn:nbn:hr:163:468557

D. Gnjilac, "Usporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku", Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, 2020. Available at: https://urn.nsk.hr/urn:nbn:hr:163:468557

Metadata
TitleUsporedba mikrokristalične celuloze, C18 te HILIC adsorbensa u svrhu pročišćavanja glikopeptida ljudskog imunoglobulina G za visokoprotočnu glikoproteomiku
Title (english)Comparison of microcrystalline cellulose, C18 and HILIC adsorbents for the purpose of purification of human immunoglobulin G glycopeptides for high-throughput glycoproteomics
AuthorDario Gnjilac
MentorGordan Lauc (mentor)
Committee memberGordan Lauc (predsjednik povjerenstva)
Committee memberLidija Bach-Rojecky (član povjerenstva)
Committee memberJasna Jablan (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Pharmacy and Biochemistry
(Department of biochemistry and molecular biology)
Zagreb
Defense date and country2020-09-10, 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
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:163:468557
Study programmeTitle: Pharmacy
Study programme type: university
Study level: integrated undergraduate and graduate
Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2021-04-30 14:32:10