RESUMO
Metaproteomics has emerged as a new, revolutionary approach to study gut microbiota functionality, but the lack of consistent studies in this field due to the great complexity of samples has prompted to search new strategies to achieve better metaproteome characterization. Some steps in sample preparation and data analysis procedures are critical for obtaining accurate results, therefore protein extraction buffers, digestion procedures and fractionation steps were tested here. Initially, two lysis buffers were used to improve protein extraction, two common digestion protocols were compared, and fractionation processes were employed at both the peptide and protein levels. The combination of these procedures resulted in five different methodologies; SDS buffer, in-gel digestion and fractionation at the peptide level provided the best results. Finally, the metaproteomics workflow was tested in a real case study with obese rats, in which a metagenomics study was previously performed. Important differences in protein levels were observed between groups that were potentially related to the taxonomical family, indicating that functional processes are modulated by the microbiota. Therefore, in addition to the necessity of combining different metaomics approaches, an optimized metaproteomics workflow such as the presented in this study is required to obtain a better understanding of the microbiota function. SIGNIFICANCE: Gut microbiota has emerged as an important factor with affects the health balance in host. To study its function new methodologies are necessary and the most appropriate one seems to be metaproteomics. The lack of studies in this field requires a deeply research in the most accurate workflow to better comprehend such complex samples. In this paper, five different methodologies have been compared, mainly in the most critical steps in classical proteomics and the methodology chosen was validated in a real case study in obese animals.