Proteomska analiza mononuklearnih celic periferne krvi pri raku dojk

magistrsko delo

Dea Simonič (Avtor), Uroš Potočnik (Mentor), Darja Arko (Komentor), Maya Petek (Komentor)

Povzetek

Rak dojk je najpogostejši rak pri ženskah in obenem najpogostejši vzrok smrti zaradi raka pri ženski populaciji. Rak te vrste za uspešno napredovanje in metastaziranje preoblikuje okoliško tkivo in ustvari lastno tumorsko mikrookolje. Slednje med drugim vključuje spremembe v celicah imunskega sistema, katerega ključna komponenta za boj proti infekcijam in prilagajanje patogenom so mononuklearne celice periferne krvi (MCPK). Posledično proteini MCPK lahko predstavljajo pomembne biomarkerje imunskih bolezni, med drugim tudi rakavih obolenj, saj je znano, da se ob nastanku raka spremeni proteom MCPK. Med naborom tehnik, s katerimi je mogoče v velikem obsegu raziskovati proteine, je zaradi svoje sposobnosti obvladovanja proteomske kompleksnosti postala razširjena tekočinska kromatografija, sklopljena s tandemsko masno spektrometrijo (LC MS/MS). V magistrski nalogi smo optimizirali metodo izolacije in priprave proteinskih vzorcev iz MCPK, ki je preprosta, učinkovita in neposredno združljiva s sistemom LC-MS/MS. Učinek optimizacije smo ocenili na podlagi števila identificiranih peptidov in proteinskih skupin v vsakem vzorcu ter na podlagi ponovljivosti metode. Najprej smo preizkusili različne sestave liznih pufrov za izolacijo proteinov in ugotovili, da je najbolj obetaven lizni pufer MSC RIPA na osnovi natrijevega deoksiholata in etilen glikola. Ker sprememba koncentracij površinsko aktivnih snovi v MSC-RIPA pufru ni pokazala izboljšanja pri identifikaciji proteinov z masnim spektrometrom, smo v protokol dodali korak frakcionacije peptidov z močno kationsko izmenjavo na različno število frakcij. Za možnost analize večjega števila vzorcev v istem času smo optimizirali tudi trajanje kromatografske metode na LC MS/MS instrumentu. Končna optimizirana metoda obsega lizo MCPK z MSC-RIPA pufrom, SCX frakcionacijo vzorcev na tri frakcije ter analizo na LC-MS/MS z 75-minutnim kromatografskim gradientom. Z optimizirano metodo smo dosegli povprečno 3 373 (s.d. ± 52) identificiranih proteinskih skupin na vzorec MCPK z metodo, ki omogoča analizo 21 vzorcev na teden. Optimizirana metoda nam je omogočila izvedbo preliminarne proteomske študije na šestih vzorcih MCPK bolnic z rakom dojk – dosegli smo povprečno 3 370 (s.d. ± 99) identificiranih proteinskih skupin na vzorec. Te smo primerjali s tremi vzorci MCPK zdravih posameznikov in izvedli analizo diferencialne ekspresije. Na podlagi opravljane analize diferencialne ekspresije na vzorcih treh kontrol, treh vzorcih bolnic z luminalnim podtipom A ter treh vzorcih bolnic z luminalnim podtipom B, smo identificirali tri proteine, ki so bolj izraženi v vzorcih MCPK bolnic z rakom dojk in bi lahko bili potencialni biomarkerji raka dojk iz periferne venske krvi. Tako optimizirana metoda bo uporabljena za študijo proteoma MCPK na večji kohorti bolnic z rakom dojk, seveda pa jo je mogoče uporabiti tudi za preučevanje drugih bolezni.

Ključne besede

proteomska analiza;MCPK;masna spektrometrija;rak dojk;magistrske naloge;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2023
Tipologija:	2.09 - Magistrsko delo
Organizacija:	UM FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[D. Simonič]
UDK:	547.963:618.19-006(043.2)
COBISS:	171068675
Št. ogledov:	81
Št. prenosov:	14
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	Proteomic analysis of periferal blood mononuclear cells in breast cancer
Sekundarni povzetek:	Breast cancer is the most common cancer in women and the leading cause of cancer death in women. Breast cancer transforms the surrounding tissue and creates its own tumour microenvironment to progress and metastasise. The tumour microenvironment includes changes in the cells of the immune system, a key component of which, in order to fight infections and adapt to pathogens, are the peripheral blood mononuclear cell (PBMC). Consequently, expression of PBMC proteins may represent important biomarkers of immune diseases, including cancer, as the PBMC proteome is known to be altered at the onset of cancer. Among the range of techniques that can be used to study proteins on a large scale, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become widespread due to its ability to handle proteomic complexity. In this thesis, we optimised a method for isolation and preparation of protein samples from PBMC that is simple, efficient and directly compatible with LC-MS/MS. The optimisation performance was evaluated with the number of peptides and protein groups identified in each sample and the reproducibility of the method. First, we tested different lysis buffer compositions for protein isolation and found that the most promising was MSC-RIPA lysis buffer, which is based on sodium deoxycholate and ethylene glycol. Since varying the surfactant concentrations in the MSC-RIPA buffer did not show an improvement in protein identification by mass spectrometry, we added a peptide fractionation step using strong cation exchange, giving different numbers of fractions. The duration of the chromatographic method on the LC-MS/MS instrument was also optimised to allow the analysis of a larger number of samples in a given time. The final optimised method consists of lysis of PBMC with MSC-RIPA buffer, SCX fractionation of the samples into three fractions and LC-MS/MS analysis with a 75-minute chromatographic gradient. The optimised method yielded an average of 3 373 (s.d. ± 52) identified protein groups per PBMC sample with a method allowing the analysis of 21 samples per week. The optimised method allowed us to perform a preliminary proteomic study on six PBMC samples from breast cancer patients, achieving an average of 3 370 (s.d. ± 99) identified protein groups per sample. These were compared with three PBMC samples from healthy individuals and differential expression analysis was performed. A comparison of three controls, three samples of patients with luminal A and three samples of patients with luminal B, showed three proteins that are more highly expressed in the PBMC samples of breast cancer patients and could be potential biomarkers of breast cancer from peripheral venous blood. This optimised method will be used to study the PBMC proteome in a larger cohort of breast cancer patients and can of course also be used to study other diseases.
Sekundarne ključne besede:	proteomic analysis;PBMC;breast cancer;mass sprectrometry;
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 (XX, 157 f.))
ID:	19863893