Project description:The data set consist of three different sources. 1) All files with ecoli_* derive from a pure culture of Escherichia coli K-12 (MG1655). 2) All files with SIHUMI_standard_* derive from a mixed culture of 8 bacteria (SIHUMIx) Anaerostipes caccae (DSMZ 14662), Bacteroides thetaiotaomicron (DSMZ 2079), Bifidobacterium longum (NCC 2705), Blautia producta (DSMZ 2950), Clostridium butyricum (DSMZ 10702), Clostridium ramosum (DSMZ 1402), Escherichia coli K-12 (MG1655) and Lactobacillus plantarum (DSMZ 20174). A standard proteomic protocol was used for purification. 3) All files with SIHUMI_small_* derive from the same bacteria culture as second source in contrast a variety of different proteomic protocols were used to enhance enrichment of small (<100 AS) Proteins. The goal of the project was to design a workflow to quickly prioritize novel protein candidates. The workflow was designed to be robust in a meta-omics context and facilitate the integration of transcriptomic and other information on a genomic level. The MS-data from the first source was used to test the workflow under well controlled conditions, namely in pure culture and near complete annotation. The workflow was used with data from the second source to see if good results can be produced in a mixed culture. To enhance the chances of finding novel proteins we incorporated the data from the third source.
Project description:Short open reading frames (sORF) have the potential to encode small proteins in the human gut microbiome, but their role in this environment is rarely understood. These small proteins, known as sORF-encoded peptides (SEP), are less than or equal to 100 amino acids in length. Using bottom-up proteomics (BUP) and top-down proteomics (TDP) approaches, we aimed to identify SEP under various growth conditions and stress exposure in Blautia producta, a key species in mediating colonization resistance and dampening gut inflammation. After applying a rigorous filtering and validation procedure, we identified 45 SEP. Our findings indicate that in contrast to previous results the production of specific SEP is not restricted to direct bacterial interactions within the microbiome but rather depends on growth and stress conditions of the environment. Top-down analysis improved identification confidence and detected a number of full-length with and without N-terminal methionine excision (NME), N- or C-terminal truncated or post-translational modified proteoforms of SEP, indicating that these are common occurrences in B. producta. These findings highlight SEP as a ubiquitous class of non-annotated polypeptides that have been overlooked as a subset of proteins in B. producta.
