Uspostavljanje protokola za analizu glikozilacije male količine imunoglobulina G iz početnog uzorka velikog volumena

master's thesis

• Title (english): Protocol development for glycosylation analysis of small amount of IgG from the initial large-volume sample
• Author: Angela Milanović
• Mentor: Gordan Lauc (mentor)
• Committee member: Gordan Lauc (predsjednik povjerenstva)
• Committee member: Tin Weitner (član povjerenstva)
• Committee member: Jasminka Krištić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2019-07-09, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Glikozilacija je ključna posttranslacijska modifikacija čiji je utjecaj na funkciju proteina vjerojatno
najbolje pokazan na primjeru imunoglobulina G (IgG). Budući je najzastupnije antitijelo u serumu
i time lako dostupan, glikozilacija IgG-a vrlo je dobro opisana, kao i promjene u zastupljenosti
pojedinih N-glikana IgG-a u raznim stanjima i bolestima, zbog čega IgG predstavlja odličan model
glikoproteina. U svrhu daljnjeg rasvjetljavanja glikozilacije, razvijene su brojne metode koje
omogućuju analizu slobodnih glikana te glikopeptida. Veliki izazov predstavlja prilagođavanje
postojećih metoda uzorcima male količine glikoproteina za čiju je analizu potrebno razviti
osjetljivije metode. Uzorci supernatanta staničnih kultura koje se ponekad koriste u istraživanju
glikozilacije često sadrže vrlo male količine IgG-a u velikim volumenima medija jer su stanični
modeli nerijetko loši proizvođači IgG-a. Obzirom da su postojeće metode za analizu glikozilacije
IgG-a uglavnom optimizirane za uzorak plazme/seruma, bilo je potrebno uspostaviti protokol za
analizu N-glikozilacije IgG-a iz početnih uzoraka male količine IgG-a u velikom volumenu,
točnije, provjeriti mogu li se postojeće metode, uz manje modifikacije i optimizacije, koristiti u
navedene svrhe. Optimiranjem pojedinih koraka uspostavljen je protokol koji se sastoji od
izolacije IgG-a afinitetnom kromatografijom na ÄKTA Avant 25 sustavu uz CIM r-Protein G disk
monolitnu kolonu, odsoljavanja eluiranog IgG-a hladnim metanolom, deglikozilacije izoliranog
IgG-a pomoću PNGaze F, obilježavanje oslobođenih glikana APTS-om, pročišćavanja glikana na
Bio-gel P-10 te analize glikana kapilarnom gel elektroforezom. Analizom uzoraka male količine
IgG-a (1 – 10 µg) u volumenu od nekoliko desetaka mililitara staničnog medija ustanovljeno je da
je osjetljivost uspostavljene metode dovoljna za analizu profila glikana porijeklom IgG-a količine
od 3 µg. Za postizanje osjetljivosti dovoljne za analizu i manjh količina IgG-a (nanogramskih),
potrebne su veće modifikacije metode poput odabira druge tehnike odsoljavanja proteina te
dodatnog pročišćavanja i mogućnosti ukoncentriravanja obilježenih glikana.

Abstract (english)

Glycosylation is the key posttranslational modification whose impact on the function of proteins
is probably best described on the example of immunoglobulin G (IgG). Since it is the most
abundant antibody in human serum, and therefore easily accessible, glycosylation of IgG is very
well described, just as presence of different N-glycans in various states and diseases, all of which
makes IgG an excellent model glycoprotein. Fast progress in glycoscience requires establishment
of high-throughput technologies for analysis of N-glycans, few of which have been developed
recently. One of the challenges is adaption of existing technologies to low-abundance
glycoprotein samples, for whose analysis development of more sensitive methods is needed.
Since cell models sometimes used for the purpose of glycosylation research are often poor
producers of IgG, samples of cell culture supernatants contain scarce amounts of IgG in big
volumes of media. Considering that existing methods are optimized for the analysis of
plasma/serum samples, it was necessary to establish a protocol for the IgG N-glycosylation
analysis from initial large-volume samples with small quantity of IgG. To be more precise, the
aim was to verify whether the existing methods, with minor modifications and optimization,
could be used for analysis of samples containing 1-10 µg of IgG in initial volumes of tens of
millilitres. Following optimization of mainly the isolation and desalting steps, APTS labelled Nglycans released form IgG isolated by affinity chromatography were analysed by capillary gel
electrophoresis. Established protocol is proved sensitive enough for analysis of glycans derived
from 3 µg of IgG. Major modifications, such as selection of another desalting technique, adding
additional purification steps and concentration of labelled glycans samples could result in greater
sensitivity increase needed for analysis of smaller amounts (nanograms) of IgG.

• Keywords: imunoglobulin G; N-glikani; uzorak velikog volumena; CGE-LIF
• Keywords (english): immunoglobulin G; N-glycans; large-volume sample; CGE-LIF
• Language: croatian
• URN:NBN: urn:nbn:hr:163:097771
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-02-14 14:23:05