De novo-assisted peptidomics helps in the study of marine carbon flux and protein degradation

De novo peptide sequencing, where the sequence of amino acids is determined directly from mass spectra rather than from comparison with theoretical spectra from a selected protein database, is a promising alternative and compliment to the database-driven approach currently used for environmental metaproteomics. When used in combination with traditional database searching, de novo-assisted peptide sequencing boosts coverage by 10% for a model axenic culture of Prochlorococcus marinus. Peptide identification in suspended and sinking particles collected from three depths (100, 265, and 1000 m) in the eastern tropical North Pacific Ocean (ETNP) was similarly enhanced using the de novo assisted approach. De-novo identified peptides included high confidence matches to proteins from taxa unanticipated to be in the relatively uncharacterized sinking particulate matter, including many from the fungus Dikarya. When Dikarya proteins were added to the database, the metaproteomic approach then identified the fungal peptides, confirming the utility of the de novo assisted approach for complex environmental samples where the corresponding metaproteomic database is imperfect. The de novo-assisted approach also helps in understanding organic carbon and nitrogen degradation in the ocean. De novo sequencing of sinking particles from 1000 identified short (<10 amino acid) length non-tryptic peptides sourced from the autotrophic bacterium Procholorococccus that were missed by the metaproteomic approach. These peptides are most likely in the process of being degraded as they sink to the interior of the ocean. De novo identified peptides at depth contain more mass modifications of amino acids relative to those at the surface, including deamidation and formylation. Deamidation has previously been hypothesized as a source of ammonium to support anammox in this region, suggesting that the de novo tool provides a molecular-level view into the processes fueling chemoautotrophy in the ETNP.