Metaproteomics of gastrointestinal microbiota of a healthy person

Crijevna mikrobiota čovjeka se sastoji od velikog broja mikroorganizama te se njihova disbioza povezuje s mnoštvom bolesti. Identifikacija tih mikroorganizama je vrlo važna kako bi se bolje rzumjela veza između bolesti i crijevne mikrobiote. Metaproteomika koja se definira kao identifikacija svih proteina iz mikrobne zajednice nekog uzorka pruža informacije o vrsti mikroorganizma. Zasniva se na digestiji proteina, sekvencioniranju peptida tandemnom spektrometrijom masa i pretragom baza podataka. Cilj ovog rada je bio usporediti utjecaj metoda za izolaciju proteina i utjecaj spekrometra masa te pretražnog programa na taksonomsku kompoziciju crijevne mikrobiote. Rezultati su uspoređeni s literaturnim podacima i s genomskom analizom. Najviše proteina i bakterijskih vrsta je određeno koristeći 4% SDS i B-PER u kombinaciji s ultrazvukom kada se analiza provodila na spektrometru masa ESI-Q-TOF i kada je korišten novorazvijeni bioinformatički program temeljen na LSI. Metagenomska analiza je rezultirala manjim brojem bakterija Bacteroidetes nego metaproteomika te se podudarala s rezultatima metaproteomike u koljenima Firmicutes i Proteobacteria. Human intestinal microbial consists of a large number of microorganisms and their dysbiosis is associated with a great number of diseases. Identification of these microorganisms is very important in order to better interrupt the connection between the disease and the intestinal microbiota. Metaproteomics, which is defined as the identification of all proteins from a sample, provides information on the species of microorganisms. The worklow consists of digestion of proteins, sequencing of the peptides by tandem mass spectrometry and database searching. The aim of this study was to compare the effect of the protein isolation method and the influence of the mass spectrometer and the bioinformatic tool on the taxonomic composition of the intestinal microbiote. The results were compared with literary data and genomic analysis. The biggest number of bacterial species was determined using 4% SDS and B-PER in combination with sonification when analyzed on ESI-Q-TOF mass spectrometer and when a newly-developed bioinformatic LSI tool was used. Metagenomic analysis resulted in a smaller number of bacteria from phyilia Bacteroidetes than metaproteomics but it had similar results in phylia Firmicutes and Proteobacteria.