Protocol optimization for feces microbiota protein isolation

U novije vrijeme proteomika mikrobiota fecesa (metaproteomika) zauzima važno područje u istraživanju patogeneze mnogih bolesti, kao i u ranoj dijagnostici. Često se u ovim istraživanjima koriste modeli organizama poput primjerice štakora, a tehnološka platforma koja postaje sve važnija za brzo, sveobuhvatno i precizno definiranje metaproteoma, odnosno ukupnog proteoma mikrobiota, je masena spektrometrija (MS). Sastav mikrobiota fecesa štakora vrlo je kompleksan i obuhvaća različite vrste bakterija, kvasaca te virusa. Stoga je u svrhu proteomskih istraživanja potrebno razvijati efikasne metode za izolaciju različitih vrsta mikrobiota što podrazumijeva i različite načine za razbijanje stanica u svrhu izolacije molekularnih sastavnica iz tih stanica, primjerice ukupnih proteina. Neke od metoda koje se koriste za izolaciju proteina iz mikrobiota, a koje su kompatibilne sa MS uključuju sonikaciju i homogenizaciju keramičkim kuglicama. Uspješnost protokola za pripremu uzorka u proteomskim istraživanjima metaproteoma ključna je za uspješnu i nedvojbenu identifikaciju. U ovom je radu stoga proveden razvoj i tehnička optimizacija protokola za izolaciju ukupnog proteinskog komplementa mikrobiota fecesa štakora. U tu svrhu koristili smo mješavinu bakterijskih kultura i kulture kvasca koja je simulirala sastav mikrobiota fecesa štakora. Stanice mikroorganizama smo razbijali uz kombinacije mehaničkih ili fizikalnih metoda zajedno s kemijskim metodama. Uspješnost pojedinih protokola za izolaciju proteina iz mikrobiota pratili smo kvantitativnim metodama, 1 dimenzionalnom (1D) gel elektroforezom te identifikacijom proteina pomoću MS koja se temelji na matricom potpomognutoj ionizaciji laserskom desorpcijom uz analizator preleta leta (MALDI-TOF MS). Također je provedena optimizacija protokola za digestiju proteina tripsinom na filtru (engl. Filter Aided Sample Preparation – FASP). Najefikasniji protokol za izolaciju proteina bio je temeljen na homogenizaciji stanica kuglicama u kombinaciji s puferom za razbijanje stanica koji sadrži kaotrope te neutralni deterdžent ili ionski deterdžent.Faecal microbiota proteomics (metaproteomics) occupies an important area in the research of many diseases pathogenesis as well as in early diagnosis. Often, in these studies, model organisms such as rats, are used. Moreover, an increasingly important technology platform for rapid, comprehensive and precise definition of the metaproteome, which is a complement of the total microbial proteome, is mass spectrometry (MS). The composition of the rat faecal microbiota is very complex and includes various types of bacteria, yeasts, and viruses. Therefore, for the purpose of proteomic research, it is necessary to develop efficient methods for isolation of different types of microorganisms, which includes different approaches for cell lysis and isolation of molecular constituents from these cells, for example, total protein. Some of the methods used for isolation of microbial proteins that are compatible with MS include sonication and homogenization by ceramic beads. Adequate protocol optimization for metaproteome analysis is crucial for successful and unambiguous identification. Therefore, this thesis presents development and technical optimization of the protocol for the isolation of the total protein complement of the faecal rat microbiota. For this purpose, we used a mix of bacterial and yeast cultures that simulated the composition of the faecal rat microbiota. Microorganisms’ cells were lysed by combinations of mechanical or physical methods and chemical methods. The success of all microbial protein isolation protocols was assessed by protein quantification methods, 1 dimensional (1D) gel electrophoresis and protein identification by MS-based technique known as matrix assisted laser desorption/ionization time of flight (MALDI-TOF MS). Filter Aided Sample Preparation (FASP) protein digestion protocol development and optimization was also performed. The most efficient protein isolation protocol was based on the homogenization of cells using ceramic beads combined with a lysis buffer containing chaotropic agents and a neutral detergent or ionic detergent