Primjena tehnologije LC-MS/MS za proteomsku analizu gingivnog tkiva: eksperimentalno istraživanje The Application of LC-MS/MS Technology for Proteomic Analysis of Gingival Tissue: a Pilot Study
نویسندگان
چکیده
Adresa za dopisivanje Ana Badovinac, dr. med. dent. i doc. dr. sc. Darko Božić Sveučilište u Zagrebu Stomatološki fakultet Zavod za parodontologiju Gundulićeva 5, HR – 10000 Zagreb, Hrvatska Tel: +3851489920 [email protected], [email protected] Sažetak Svrha: Željelo se pronaći optimalne uvjete i metode za identifikaciju maksimalnog broja proteina u uzorcima tkiva gingive pacijentice s agresivnim parodontitisom. Pritom smo se koristili tehnologijom Label-free kvantifikacije, tekućinske kromatografije i spektrometrije masa (LC –MS/MS), te usporedili proteom zdravih i bolesnih uzoraka tkiva gingive. Materijali i metode: Četiri uzorka gingivnog tkiva (dva zdrava i dva bolesna) uzeta su od pacijentice, inače nepušačice, koja boluje od teškog oblika generaliziranog agresivnog parodontitisa. Proteini tkivnog lizata razdvojeni su 1D gel-elektroforezom nakon koje je slijedila digestija u gelu te mjerenje proteina nanoljestvicom HPLC-sistema preko nanoelektro spreja, ionizacijskog izvora i spektrometra mase. Dobiveni podaci obrađeni su u programu MaxQuant. Rezultati: Label-free kvantifikacija i LC-MS/MS analiza, zajedno s pripremom uzoraka, 1D gel-elektroforezom i digestijom u gelu pokazali su se kao učinkovita metoda za analizu proteoma gingivnog tkiva. Ukupan broj identificiranih i kvantificiranih proteina iznosio je 2429. Proteini koji su pokazali pojačanu ekspresiju u tkivu zahvaćenom agresivnim parodontitisom bili su: imunoglobulinski gama-1 lanac regije C, imunoglobulinski kapa lanac regije C i imunoglobulinski gama-3 lanac regije C (Ig gamma-1 chain C region, Ig kappa chain C region i Ig gamma-3 chain C region), a desmoplakin, aneksini, proteini S100-A8/A9 i keratini pokazali su sniženu ekspresiju. Zaključak: Ovo eksperimentalno istraživanje omogućuje novi uvid u proteomski profil zdravog i bolesnog gingivnog tkiva, što bi moglo pridonijeti napretku u dijagnostici i prognozi te boljem razumijevanju patogeneze agresivnog parodontitisa. Tehnologija LCMS/MS pokazala se učinkovitom kad je riječ o proteomskoj analizi gingivnog tkiva. Ključne riječi parodontitis, agresivni; gingiva; proteomika; spektrometrija masa; biopsija; bjelančevine vezane na mikrotubule; imunoglobulin kappa lanci; dezmoplakini; aneksini; keratini; S100 bjelančevine 1 Zavod za parodontologiju Stomatološkog fakulteta Sveučilišta u Zagrebu i Klinički zavod za parodontologiju Kliničkog bolničkog centra Zagreb, Zagreb, Hrvatska Department of Periodontology, School of Dental Medicine, Zagreb, and Clinical Department of Periodontology, Clinical Hospital Center Zagreb, Zagreb, Croatia 2 Centar za translacijska i klinička istraživanja Medicinskog fakulteta Sveučilišta u Zagrebu, Hrvatska Center for Translational and Clinical Research, School of Medicine, Zagreb, Croatia
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