Protocol optimization for protein isolation from FaDu cell line fractions for proteomic analysis using mass spectrometry

Rak glave in vratu (angl. Head and Neck Cancer, HNC) predstavlja raznoliko skupino rakov, med katere uvrščamo rake ustne in nosne votline, grla in žrela. Kljub napredku pri razumevanju genetskih dejavnikov, dejavnikov okolja, molekularnih in celičnih mehanizmov, ki sodelujejo pri nastanku raka glave in vratu, so rezultati trenutno odobrenih načinov zdravljenja, po stopnji ponovitve bolezni in umrljivosti, zelo zaskrbljujoči. Po številu novih primerov in po številu smrti na letni ravni se rak glave in vratu uvršča na šesto mesto najpogostejših rakov na svetu. Iz tega razloga sta nadaljnja molekularna karakterizacija kancerogeneze in novih molekulskih terapevtskih tarč ključnega pomena. Cilj magistrske naloge je bil določiti proteom posameznih celičnih frakcij ploščatoceličnega karcinoma glave in vratu. Optimizirali smo postopek celične frakcionacije celične linije FaDu na osnovi različno močnih surfaktantov in diferencialnega centrifugiranja, s katerim smo celične beljakovine ločili na citosolne, jedrne in frakcijo, ki je vsebovala beljakovine membranskih organelov vključno s proteini celične membrane. Učinkovitost subcelične frakcionacije smo preverili z metodo western blot z uporabo ustreznih proteinskih označevalcev. Proteomsko analizo posameznih frakcij smo opravili z masno spektrometrijo visoke ločljivosti in načinom za neodvisno pridobivanje spektralnih podatkov. Identificirali smo 5273 beljakovin v treh celičnih frakcijah, od tega smo 2730 beljakovinam določili ustrezno celično lokacijo z nenadzorovanim strojnim učenjem s programom MetaMass. V nalogi smo kot prvi na celičnem modelu celične linije FaDu določili proteom jedrne in citosolne frakcije ter frakcije membranskih organelov ploščatoceličnega karcinoma glave in vratu. V nadaljevanju bo potrebno klinično in terapevtsko uporabnost okarakteriziranih beljakovin še potrditi.Head and Neck Cancer (HNC) is a diverse group of cancers, including cancers of the oral and nasal cavities, the larynx, and the pharynx. Despite advances in the understanding of genetic and environmental risk factors as well as the molecular and cellular mechanisms involved in the development of HNC, the clinical outcomes of current therapies in terms of disease relapse and mortality rates are of great concern, as HNC ranks as the sixth most common cancer worldwide. To this end, further molecular characterization of HNC carcinogenesis and new molecular therapeutic targets are of crucial importance. In this thesis, to analyze the proteome of subcellular fractions of head and neck squamous cell carcinoma, optimizing cell fractionation of the FaDu cell line was based on surfactants of increasing stringency and differential centrifugation to isolate cytosolic and nuclear fractions and a fraction containing membrane organelles, including the plasma membrane. The efficiency of subcellular fractionation was verified by western blot. Proteome analysis of crude protein fractions was done by data-independent acquisition with high resolution mass spectrometry, using label-free quantification to determine protein abundance. A total of 5273 proteins were identified in the three cell fractions, of which 2730 proteins were assigned the corresponding cellular location by unsupervised machine learning with MetaMass. In this work, we are the first to analyse the proteome of cytosolic, nuclear and membrane organelle fractions in the head and neck squamous cell carcinoma FaDu cell model. Further studies are needed to evaluate the efficacy of identified proteins as potential clinical and therapeutic targets