A peptide database from human feces to foster the discovery of novel bioactive peptides

Fecal microbiota transplantation (FMT) is known for its high efficacy in addressing gastrointestinal, metabolic, and neurological disorders, as well as various types of cancer.However, sterile stool filtrates (SSF) have also been proven effective in the treatment of a variety of diseases 1,2 , posing a lower risk for bacterial infection than FMT treatment, particularly in immunocompromised patients 1 .SSF consists mainly of microbiota-derived polypeptides and gut metabolites, but the nature and identity of antimicrobial or immunomodulatory factors are largely unknown.We have previously shown that body fluid filtrates are a rich source of novel anticancer and antimicrobial peptides with diagnostic and therapeutic potential 4 .Therefore, in the present work, we focused on the generation and analysis of a peptide bank from human feces for the identification of novel antimicrobial, immunomodulatory, and anticancer peptides, either using in silico approaches or bioassay-guided isolation and characterization of specific peptide components.A liquid sample (2L) of human feces from healthy donors was filtered through a membrane with a cut-off of 30 kDa.The filtered sample was subjected to reversed-phase HPLC; 36 fractions were collected and stored at -80C until further use.A 0.1 mL aliquot of each fraction was used to form six small pools of adjacent fractions for sample preparation and mass spectrometry.All pools of fractions were processed for heme group removal by either cold acetone extraction or C18-SPE and then subjected to sequencing analysis by LC-MSMS and bioinformatics.Several hundred peptides were identified as bacterial or human protein fragments, as well as many de novo-sequenced peptides.An in silico approach was also used to predict antimicrobial peptides among them.We expect that our methodology, which includes the generation of the human feces peptide bank, sample processing, and bioinformatics analyses, will facilitate the discovery of novel human peptides of therapeutic interest.