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Protein cycling in the eastern tropical North Pacific oxygen deficient zone: a de novo-discovery peptidomic approach

ABSTRACT: Peptides and proteins were identified using a novel de novo-discovery approach in suspended and sinking organic particles from the eastern tropical North Pacific and in a culture of a dominant autotroph from the region, the cyanobacterium Prochlorococcus. De novo peptide sequencing, where the sequence of amino acids is determined directly from mass spectra rather than from comparison to theoretical spectra from a selected sequence database, was found to be a useful tool for discovery of peptides present in a sample but not initially included in the search database. Iterative de novo-informed database search results suggested the presence of fungal peptides and proteins in deep sinking particles, consistent with growing evidence that fungi play an important role in degradation of sinking material in the ocean. The de novo-discovery approach also allowed the tracking of modified autotrophic cyanobacterial peptides to the deep sea, where they contributed 0.63% of the phylum-level identifiable peptide pool in a bathymetric sediment trap sample. Overall, the amino acid composition of the peptides in the sinking material showed little change with depth, consistent with earlier observations of bulk organic matter and/or amino acid composition during the early stages of degradation. However, we identified an abundance of modified amino acids in sinking and suspended particles, including high levels of deamidation, suggesting that partial degradation of protein could potentially fuel observed anammox and contribute to observed pool of refractory organic nitrogen. We also observe methylation of arginine, which has previously been shown to slow degradation of peptides in seawater. Our results demonstrate several examples how de novo-discovery allows for a deeper evaluation of proteins and peptides in environmental systems undergoing degradation.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Environmental Samples <unclassified

SUBMITTER: Richard Keil

LAB HEAD: Richard Keil

PROVIDER: PXD023187 | Pride | 2022-01-10

REPOSITORIES: Pride

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Project description:Peptides were identified during degradation of the diatom Thalassiosira weissflogii with and without the use of the enzyme trypsin. This allows comparison of protein degradation occurring within the experiment (no trypsin added; peptides already present due to in situ degradation) and the protein still available for future degradation (peptides released from protein when trypsin is added during analysis). Over the 12-day degradation experiment 31% of the particulate organic carbon was depleted and there was no preferential degradation of the overall protein pool. However, there was distinct differentiation in the cellular location, secondary structure and modifications of the peptides that were either degraded or remained. During the initial period of rapid algal decay and bacterial growth, intracellular components from the cytoplasm were selectively consumed, resulting in the accumulation of membrane-associated proteins and peptides in the detrital pool. Accompanying this was an increase in the importance of membrane-bound ɑ-helix motifs. At the end of the 12 day experiment, the natural bacterial assemblage was focusing on degradation of membrane-associated proteins, which was the dominant source of peptides in both the residual pool (released by trypsin) and the actively degraded pool (no trypsin added). Methylated arginine, a post-translationally modified amino acid that is produced within the diatom prior to senescence, is found in high amounts within the detrital peptide pool, suggesting a link between in-cell modification and resistance to immediate degradation. Another modification - asparagine deamidation - appears to occur during degradation and deamidated peptides also accumulate within the detritus. The bacterial community decomposing the algal material was rich in proteobacteria, and employed a growth approach focusing on accumulation of solubilized material across their membranes and on DNA replication. At this early stage of diagenesis, no changes in bulk amino acids (THAA) were observed, yet a peptidomic approach allowed us to observe the differential changes in diatom protein preservation by discriminating between intracellular location, secondary structure, and modifications status.

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Protein cycling in the eastern tropical North Pacific oxygen deficient zone: a de novo-discovery peptidomic approach

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