Optimization of a protocol for soil analysis through mass spectrometry-based proteomics and taxonomic and functional analysis of microbial communities to assess soil health status

In recent years, it has been increasingly evident that the biological and microbial community in soils are markers for understanding the soil health and quality. Indeed, it was confirmed that the type of crop, as well as the type of treatment of the soil, as well as the type of fertilization used, deeply influences the composition of the microbial community of the soil. Particularly dangerous for the public health, is the presence of ARGs (antibiotic resistance genes) some of which are the result of natural evolutionary biological processes, often due to spontaneous gene mutations or gene transfer. Most, however, are man-made coming from wastewater, intensive livestock farms or places where antibiotics are produced. Having said that, the main goal of this study was to test different published protein extraction protocols that would allow to reliably, accurately, and in a short time frame to analyse the microbial communities in soils from agricultural origin. In this study, six different soil samples, coming from different farms around Greifswald - the exact location of which is unknown for privacy reasons - were investigated using three different protein extraction protocols and a variant of one of them, in order to find a reliable, reproducible and efficient protocol. During the study, high variations in number of proteins were detected among the different protocols. At the end of this stage, the two best protocols in term of number of identified protein groups (B and B2) were further analysed, using a longer gradient LC-MS, to study whether the quality of the spectra obtained would improve. After the information obtained using normal and longer gradient, the data were further submitted to a series of bioinformatic analyses, in order to have an overview of what is really contained in the soil samples under study. But, given the thousands of proteins detected and the amount of data obtained, and as this is a master's thesis project, mainly focused on finding a suitable protocol for protein extraction from soils (that can be exploited by other researchers/students afterwards), only one taxonomic analysis was carried out on the identified phyla and one functional analysis concerning antibiotic resistance. The final results showed a very similar composition in phyla among the samples, both in different protocols and in different samples. A peat sample was also analysed, doing two replications and using B and B2 protocols, which confirmed the type of result and reproducibility between the protocols. Instead, the functional analysis, regarding the antibiotic resistances, showed that the main ARGs are conserved between samples, but their relative abundance can differ greatly between different samples, and in some cases, even between the same sample, using two different protocols, these results, basically, further confirmed that ARGs and resistance-associated proteins are widespread throughout the environment. Further investigations might reveal more differences about the taxonomy and the ARGs composition among the samples and, of course, among B and B2 protocols, indeed, even if they gave excellent results, they could always be improved.