Protein Extraction and Fractionation

To reduce the complexity of the protein extract, the same 1D gel fractionation approach was carried out as described in [13 (link)]. The 37 kDa and the 75 kDa band of a prestained marker (Precision Plus^™ Dual Color, Biorad) was used to define the height for cutting the lane into three fractions. Only the middle part (~37–75 kDa), containing the abundant metaproteome (AMP) was subsequently analysed. In order to control for possible run differences impairing reproducible cutting, one of the samples was run on all of the gels. Gel pieces were washed and proteins reduced, alkylated, and tryptically digested (per sample 200 ng of trypsin sequencing grade, Promega) overnight similarly as described in [23 (link)]. Following extraction from the gel-pieces, peptides were vacuum dried and stored at -20°C until used. Dried peptides were resolved in 2% formic acid and filtered through glass microfiber filters (Whatman; grade GF/C) for removal of gel particles. The peptide elutes were desalted by using OMIX tips (Agilent Technologies). After aspiring the samples 15 times, the peptides were eluted with 60% acetonitrile/2% formic acid, dried and reconstituted with 1% formic acid prior to LC-MS/MS.