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Title:Uporaba selektivnih membran za pripravo proteinskih vzorcev krvne plazme za določanje citokinov z masno spektrometrijo : diplomsko delo univerzitetnega študijskega programa I. stopnje
Authors:ID Zorko, Katja (Author)
ID Potočnik, Uroš (Mentor) More about this mentor... New window
ID Petek, Maya (Comentor)
Files:.pdf UN_Zorko_Katja_2023.pdf (29,39 MB)
MD5: BD42CD6DC2E934C4CABA477C28E764F6
 
Language:Slovenian
Work type:Bachelor thesis/paper
Typology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:Imunski sistem je sistem celic, kemičnih signalov in bioloških procesov, ki ščitijo organizem pred tujimi antigeni, kot so mikrobi, virusi, rakave celice in toksini. Prirojena imunost je obrambni mehanizem, ki se sproži takoj ali v nekaj urah po srečanju z antigenom in nima imunskega spomina. Glavna naloga prirojene imunosti je hitro pridobivanje citokinov, majhnih signalnih proteinov, ki sodelujejo pri celični komunikaciji in imunskih odzivih na mestih okužbe in vnetja. Glede na izjemno nizko koncentracijo citokinov v krvni plazmi le-te z masno spektrometrijo težko določimo. Signali citokinov se namreč izgubijo med močnejšimi signali visoko koncentriranih proteinov kot so albumin in imunoglobulini. Ker pa so ti proteini bistveno večji smo jih iz vzorcev odstranili z uporabo selektivnih membranskih filtrov, ki so prehodni samo za proteine do določene velikosti. Namen diplomske naloge je bila optimizacija postopka za pripravo proteinskih vzorcev krvne plazme za določanje citokinov z masno spektrometrijo. Uporabili smo dva postopka za izolacijo proteinov: uporaba selektivnih membranskih filtrov Amicon in gelska elektroforeza (SDS-PAGE). Sledili sta identifikacija in relativna kvantifikacija citokinov v krvni plazmi z metodo nHPLC-HRMS. Uporaba ultrafiltracijskih filtrov Amicon velja za enostavno in zanesljivo metodo koncentracije beljakovin. Med seboj smo primerjali delovanje filtrov z različnimi velikostmi por, ki prepuščajo proteine do velikosti 30 kDa, 10 kDa in 3 kDa. Filtri so pokazali primerljive rezultate med eksperimenti, vendar pa med njihovo uporabo ni bilo opaznih bistvenih razlik. SDS-PAGE je tehnika za ločevanje beljakovin glede na njihovo velikost s pomočjo ustvarjenega električnega polja. Velja za zahtevnejšo in zamudnejšo metodo izolacije proteinov. Pri določanju citokinov se je gelska elektroforeza izkazala za neuspešno, saj je bila koncentracija proteinov po filtraciji premajhna za uspešno vizualizacijo na gelu.
Keywords:citokini, imunski odziv, nHPLC-HRMS, ultrafiltracijski filtri Amicon, SDS-PAGE, masna spektrometrija
Place of publishing:Maribor
Place of performance:Maribor
Publisher:[K. Zorko]
Year of publishing:2023
Number of pages:1 spletni vir (1 datoteka PDF (XIV, 72 f.))
PID:20.500.12556/DKUM-83909 New window
UDC:543.51:615.382(043.2)
COBISS.SI-ID:147501315 New window
Publication date in DKUM:24.03.2023
Views:992
Downloads:90
Metadata:XML DC-XML DC-RDF
Categories:KTFMB - FKKT
:
ZORKO, Katja, 2023, Uporaba selektivnih membran za pripravo proteinskih vzorcev krvne plazme za določanje citokinov z masno spektrometrijo : diplomsko delo univerzitetnega študijskega programa I. stopnje [online]. Bachelor’s thesis. Maribor : K. Zorko. [Accessed 26 April 2025]. Retrieved from: https://dk.um.si/IzpisGradiva.php?lang=eng&id=83909
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Licences

License:CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Link:http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:09.03.2023

Secondary language

Language:English
Title:Using size-selective membranes to prepare blood plasma proteins for cytokine level measurement using mass spectrometry
Abstract:The immune system is a system of cells, chemical signals and biological processes that protect the body against foreign antigens such as microbes, viruses, cancer cells and toxins. Innate immunity is a defense mechanism that is triggered immediately or within a few hours after encountering an antigen and has no immune memory. A major task of innate immunity is the rapid production of cytokines, small signaling proteins involved in cell communication and immune responses at sites of infection and inflammation. Considering the extremely low concentration of cytokines in blood plasma, it is difficult to determine them by mass spectrometry. The signal of proteins with a lower concentration is lost among the stronger signals of highly concentrated proteins such as albumin and immunoglobulins. However, since these proteins are much larger we can remove them from the samples using selective membrane filters that are only permeable for proteins up to a certain size. The aim of the thesis was to optimize the procedure for the preparation of protein samples of blood plasma for the determination of cytokines by mass spectrometry. We used two procedures for protein isolation: Amicon selective membrane filters and gel electrophoresis (SDS-PAGE). This was followed by the identification and relative quantification of cytokines in blood plasma using the nHPLC-HRMS method. The use of Amicon ultrafiltration filters is considered a simple and reliable method of protein concentration and separation of components in samples according to their molecular weight. We compared the performance of filters with different pore sizes (30 kDa, 10 kDa and 3 kDa) that allow particular proteins to pass through. Despite the fact that the filters showed comparable results between experiments, no major changes were observed between the use of 30 kDa, 10 kDa and 3 kDa Amicon filters. SDS-PAGE is a technique for separating proteins according to their size using a generated electric field. It is considered a more demanding and time-consuming method of protein isolation. Gel electrophoresis has proved to be unsuccessful in the determination of cytokines. The protein concentration after filtration was too low for successful visualization of smaller proteins on the gel.
Keywords:cytokines, immune response, nHPLC-HRMS, ultrafiltration Amicon filters, SDS-PAGE, mass spectrometry


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