Naslov
Evaluation of sample preparation protocols for shotgun metaproteome characterization

Autori
Rešetar, Dina ; Karner, Dubravka ; Sokol, Filip ; Kraljević Pavelić, Sandra

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, ostalo, stručni

Skup
Godišnja skupština Hrvatskog društva za proteomiku

Mjesto i datum
Rijeka, Hrvatska, 17.11.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
shotgun proteomics, metaproteome, LC-MS/MS

Sažetak
Extensive microbiota studies driven by next- generation sequencing approaches slightly illuminated vast complexity, functions and role of microbiota in human diseases pathogenesis. Nevertheless, this studies underlined the importance of revealing complex host-microbiota interactions and introduced metaproteomic shotgun -based functional analysis into the field. Efficient and reproducible sample preparation is a prerequisite for robust and sensitive bottom-up proteomic workflow. However, this is a challenging task when complex microbiota samples, with high variability of cell structure and cellular organization are analyzed. In the presented study an independent comparison of 8 cell lysis and protein extraction protocols including, ultrasonic, bead-beating and freeze-thawing homogenization together with different buffer/detergent systems were evaluated. As a sample we have chosen three cell types namely, Gram–positive, Gram–negative bacteria and Saccharomyces cerevisiae yeast, that will allow more general conclusions to be made regarding the applicability of the sample preparation procedures. Furthermore, an optimized protocol should maximize total protein extraction yield and avoid a selective depletion of species with higher resistance to lysis. Such optimized experimental setting will be used in rat stool metaproteom characterization study.

Izvorni jezik
Engleski

Znanstvena područja
Interdisciplinarne prirodne znanosti, Biotehnologija, Interdisciplinarne biotehničke znanosti

POVEZANOST RADA