magistrsko delo
Doroteja Golob (Avtor), Uroš Potočnik (Mentor), Maya Petek (Komentor), Marina Marič (Komentor)

Povzetek

Dinamična fosforilacija in defosforilacija proteinov sta esencialna regulatorna mehanizma, ki regulirata večino celične fiziologije, zato ni presenetljivo, da so motnje v njunem delovanju pogosto vzrok za nastanek in napredovanje številnih bolezni. Fosfoproteomika je biološko pomembno področje, ki za analizo fosfoproteinov pogosto uporablja tekočinsko kromatografijo, sklopljeno s tandemsko masno spektrometrijo (LC MS/MS). Pred analizo z LC-MS/MS je ključnega pomena obogatitev fosforiliranih peptidov, ki zviša relativno količino ciljnih molekul in s tem poveča občutljivost metode. V magistrski nalogi smo optimizirali metodo obogatitve fosforiliranih peptidov za analizo z masno spektrometrijo, pri čemer smo izhajali iz nezadovoljivega protokola proizvajalca. Na voljo smo imeli dva obogatitvena materiala: PureCube Fe-NTA MagBeads in PureCube Ti-NTA MagBeads, katerih kapaciteto smo primerjali. Ugotovili smo, da je v naši izvedbi eksperimenta veliko obetavnejša obogatitev s Ti-NTA magnetnimi kroglicami, ki so že na začetku optimizacije ponujale zadovoljiv delež obogatenih fosfopeptidov, medtem ko bi za uporabo Fe-NTA morali bistveno spremeniti sestavo vezavnega pufra. Optimizirali smo razmerje med količino Ti-NTA in maso vhodnih peptidov ter delež glikolne kisline (GA) v vezavnem pufru. Rezultati kažejo, da z večjo količino afinitetnega materiala obogatimo več fosforiliranih peptidov, brez zmanjšanega deleža le-teh v vzorcu, zato smo kot optimalno določili dvakratno količino Ti-NTA glede na prvotni protokol. Nadalje smo ugotovili, da dodatek GA občutno vpliva na specifičnost vezave, vendar večji delež le-te hkrati obogati manj fosfopeptidov. Kompromis med zvišanjem specifičnosti in večjim številom fosfopeptidov smo tako dosegli pri uporabi vezavnega pufra z 0,2 % deležem GA. Na podlagi alternativnih protokolov in teorije smo prilagodili tudi inkubacijski čas vezave in elucije fosfopeptidov. Nadalje smo ugotavljali minimalno maso vhodnih peptidov, iz katere še dobimo zanesljivo kvantifikacijo fosfopeptidov. Ugotovili smo, da je ta lahko veliko nižja kot navaja proizvajalec; minimalno maso smo postavili pri 50 g, saj nižje mase dajejo zelo variabilne in s tem nezanesljive rezultate. Optimizirano metodo smo testirali na peptidih iz celic, gojenih v različnih pogojih. Analizirali smo fosfoproteom celic, tretiranih s kemoterapevtikom doksorubicin, napram celicam v normalnih rastnih pogojih. Statistična analiza je pokazala, da se je ob izpostavljenosti doksorubicinu količina številinih fosforiliranih peptidov statistično značilno povečala. Uporabljen masni spektrometer se je izkazal za dovolj občutljivega, da te spremembe zazna, optimizirana metoda pa kot učinkovita. Metoda je tako primerna za proučevanje fosfoproteoma, ko imamo opravka z manjšim številom vzorcev, pred širšo uporabo pa bo potrebna dodatna optimizacija.

Ključne besede

proteomika;fosforilacija;obogatitev fosfopeptidov;masna spektrometrija;magistrske naloge;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UM FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [D. Golob]
UDK: 543.645.6:543.51(043.3)
COBISS: 208534275 Povezava se bo odprla v novem oknu
Št. ogledov: 21
Št. prenosov: 8
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Method optimisation for phosphopeptide enrichment for analysis with mass spectrometry
Sekundarni povzetek: Protein phosphorylation and dephosphorylation are essential regulatory mechanisms that control most aspects of cellular physiology. Therefore, it is not surprising that any dysregulation of these mechanisms is often responsible for disease onset and progression. Phosphoproteomics is a biologically significant field which frequently uses liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the analysis of phosphoproteins. A crucial step prior to LC-MS/MS analysis is enrichment of phosphorylated peptides, which increases the relative amount of target molecules, thereby enhancing sensitivity of the method. In the master's thesis we have optimised the phosphopeptide enrichment method for analysis with mass spectrometry, starting from an unsatisfactory protocol provided by the manufacturer. We compared the capacities of two available enrichment materials: PureCube Fe-NTA MagBeads in PureCube Ti-NTA MagBeads. Under our experimental conditions, we found that enrichment was much more promising using Ti-NTA magnetic beads, which offered high percentage of phosphopeptides from the start of the optimisation. In contrast, the use of Fe NTA in the future experiments would require significant changes to the composition of the binding buffer. We optimised the ratio between the amount of Ti-NTA and the mass of input peptides, as well as the proportion of glycolic acid (GA) in the binding buffer. The results show that using a larger amount of affinity material enriches more phosphorylated peptides without reducing their abundance in the enriched sample. Therefore we determined that double the amount of Ti-NTA, compared to the original protocol, is optimal. Moreover, we found that while adding small proportions of GA significantly enhances binding specificity, higher proportions simultaneously enrich fewer phosphopeptides. We achieved a balance between specificity enhancement and higher yields of enriched phosphopeptides by using a binding buffer with 0,2 % GA. Based on alternative protocols and literature, we also adjusted the incubation time for the binding and elution of phosphopeptides. Additionally, we have determined the minimum mass of input peptides required for reliable quantification. We found that the input mass of peptides can be much lower than stated by the manufacturer; we set the minimum mass at 50 g, as lower masses yield highly variable and unreliable results. The optimised method was also tested on peptides from cells grown under different conditions. We analysed the phosphoproteome of cells exposed to chemotherapeutic agent doxorubicin, compared to cells in normal growth conditions. Statistical analysis revealed statistically significant increase in the amount of several phosphorylated peptides upon exposure to doxorubicin. We conclude that the mass spectrometer used is sensitive enough to detect these changes, and the optimised method proved to be effective. Thereby, the method is suitable for studying the phosphoproteome with a smaller set of samples, though further optimisation will be needed before broader application.
Sekundarne ključne besede: proteomics;phosphorylation;phosphopeptide enrichment;mass spectrometry;
Vrsta dela (COBISS): Magistrsko delo/naloga
Komentar na gradivo: Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Strani: 1 spletni vir (1 datoteka PDF (XI, 73 f.))
ID: 24822337