Ocean metaproteomics provides valuable insights into the structure and function of marine microbial communities. Yet, ocean samples are challenging due to their extensive biological diversity that results in a very large number of peptides with a large dynamic range. This study characterized the capabilities of data independent acquisition (DIA) mode for use in ocean metaproteomic samples. Spectral libraries were constructed from discovered peptides and proteins using machine learning algorithms to remove incorporation of false positives in the libraries. When compared with 1-dimensional and 2-dimensional data dependent acquisition analyses (DDA), DIA outperformed DDA both with and without gas phase fractionation. We found that larger discovered protein spectral libraries performed better, regardless of the geographic distance between where samples were collected for library generation and where the test samples were collected. Moreover, the spectral library containing all unique proteins present in the Ocean Protein Portal outperformed smaller libraries generated from individual sampling campaigns. However, a spectral library constructed from all open reading frames in a metagenome was found to be too large to be workable, resulting in low peptide identifications due to challenges maintaining a low false discovery rate with such a large database size. Given sufficient sequencing depth and validation studies, spectral libraries generated from previously discovered proteins can serve as a community resource, saving resequencing efforts. The spectral libraries generated in this study are available at the Ocean Protein Portal for this purpose.

This manuscript reports on a robot called Clio that we developed to facilitate basin-scale studies of ocean microbial communities and their biochemistry, to better understand how marine microorganisms regulate ocean and Earth system environmental cycles. Clio is designed to facilitate global-scale studies of ocean biochemistry, to move vertically through the water column with high precision, and specifically to return sensor data and samples from large swaths of the ocean ranging in depths from the surface to 6,000 m. Clio is capable of flexible, precise vertical motion that few other ocean robots can perform, and none to our knowledge over this depth range. We tested Clio extensively over several years, six cruises, and 26 dives, it is now fully operational and this manuscript describes all that we did to convince ourselves this was so. In June 2019, it completed its first large-scale ocean survey, and for which this manuscript will be the first data presentation.