engleski

Evaluation of protein extraction protocols for shotgun metaproteome characterization

Background: Efficient and robust protein extraction protocols are prerequisite however, still a challenging task when samples with higher variability of cell structure and cellular organization have to be analysed. Indeed, optimized protocols maximize total protein extraction yield and prevent selective depletion of species with higher resistance to cell lysis. Methods: Altogether 8 protein extraction protocols including, ultrasonic, bead-beating and freeze-thawing with different buffer/detergent systems were evaluated independently on a microbial community (MC) comprising of 3 Gram–positive (GPB), 2 Gram– negative (GNB) bacteria and Saccharomyces cerevisiae. Protein extraction efficacy comparison was conducted using (1) total protein quantification assay, (2) SDS-PAGE and (3) nano-LC-MS/MS approach after filter-aided sample preparation. Protein identification was performed using PLGS while for Gene Ontology protein annotation STRAP was employed. Results: SDS or urea buffer in combination with bead beating protocols resulted with the two highest protein yields. The same protocols did not result with the highest number of protein identifications but, almost equal representation of GPB, GNB and yeast proteins was reached for urea protocol. Interestingly, for other protocols were mechanical cell disruption was implemented, increased GNB protein identifications were noticed among which two sonication protocols resulted with the highest number of protein identification. For the same protocols protein yields remained low. Vast majority of identified proteins were cytoplasmic or ribosomal, involved in cellular processes and with binding, catalytic or structural molecule activity.

shotgun proteomics, metaproteomics, sample preparation, protein extraction, microbiota

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