LAUSR

Metaproteomics represent an important tool for the taxonomic and functional investigation of microbial communities in humans, environment, and technical applications. Due to the high complexity of the microbial communities, protein, and peptide fractionation is applied to improve the characterization of taxonomic and functional composition of microbial communities. In order to target scientific questions regarding taxonomic and functional composition adequately, a tradeoff between the number of fractions analyzed and the required depth of information has to be found. Two samples of a biogas plant were analyzed by either single LC‐MS/MS measurement (1D) or LC‐MS/MS measurements of fractions obtained after SDS‐PAGE (2D) separation. Fractionation with SDS‐PAGE increased the number of identified spectra by 273%, the number of peptides by 95%, and the number of metaproteins by 59%. Rarefaction plots of species and metaproteins against identified spectra showed that 2D separation was sufficient to identify most microbial families but not all metaproteins. More reliable quantitative comparison could be achieved with 2D. 1D separation enabled high‐throughput analysis of samples, however, depth in functional descriptions and reliability of quantification were lost. Nevertheless, the proteotyping of multiple samples was still possible. 2D separations provided more reliable quantitative data combined with a deeper insight into the taxonomic and functional composition of the microbial communities. Regarding taxonomic and functional composition, metaproteomics based on 2D is just the tip of an iceberg.