EAGER: Marine biopolymers as tracers of major biogeochemical processes: Using proteomics and antibody-sensor technology

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Recent advances in proteomics, biomarkers and biosensor technology sciences enable new approaches to understanding major biogeochemical processes. This project will examine the physicochemical reactivity of a model protein "RuBisCO" in seawater, and will quantify RuBisCO along ocean transect Line P (48°39.0' N, 126°40.0' W to 50°00' N, 145°00' W) in the North Pacific Ocean. The project will use two independent methods that complement and validate each other: immune-sensors and multiple-reaction monitoring (MRM) mass spectrometry.Intellectual Merit: Chemical analyses have shown that a significant fraction of dissolved organic matter (DOM) in the ocean is in the form of proteins. Proteins are a rich source of biological information and their amino acid sequence provides a direct link to the coding DNA of an organism. Identification of proteins in DOM opens a window to understanding the complex sources and dynamics of biopolymeric material. Furthermore, proteins represent the catalytic potential and reactivity of an organism and, collectively, of an ecosystem. Most importantly, the information also provides a direct link between biological and phylogenetic presence and biogeochemistry. The distributions of proteins that are specific to major processes (e.g., RuBisCO to carbon fixation, nitrogenase to diazotrophy) can be used to infer information that has been hidden until now. The PIs have recently demonstrated the deep distribution of RubisCO in the North Pacific and discovered this enzyme to be at high concentrations throughout the water column (to depths 3000 m) underlying highly productive equatorial and subpolar systems, and low concentrations under the oligotrophic subtropical gyre. This single protein represents ~2% of the largely unidentified dissolved organic nitrogen pool at depths 1000 m. The deep distribution of RuBisCO shows that hydrographic fronts in the surface ocean affect the distribution of recently-produced organic matter thousands of meters below the ocean surface, and the enzyme traces the transport of deep organic matter by the deep ocean circulation away from regions of input. These findings suggest that the identification and quantification of additional biomarker proteins will provide a powerful approach to understanding the associated biogeochemical pathways. While potentially transformative, the protocols need to be explored and validated. In this study , the PIs will compare and validate their previous findings by understanding the physicochemical reactivity of RuBisCO (and therefore other proteins) in seawater samples in order to fully interpret and exploit the information held in protein distributions; and they will quantify RuBisCO at Line P in fresh samples using two independent and different methods that complement and validate each other: immunosensors and multiple-reaction monitoring (MRM) mass spectrometry in a coastal to open ocean transect.Broader Impacts:Proteomics is in its infancy in the aquatic sciences and this research will help drive its development.Merging the sciences of proteomics with biogeochemistry will leverage both. The Sorcerer II Global Ocean Sampling (GOS) expedition predicted more than six million proteins in the GOS database, almost twice the number of proteins present in the current databases, adding an enormous diversity to the known protein families. Using immunosensors coupled with S/MRM proteomics will provide information on the cycling of RuBisCO as an actual biopolymers in the DOM pool. Current methods use hydrolysis which solely examines single amino acids but erases the chemical history of the proteins and the link between biology, phylogenetic presence, and biogeochemistry. Protein immunosensors will allow in situ tracking of marine proteins, their biosynthesis, transformation, and degradation in unconcentrated seawater. Furthermore, understanding the formation, degradation, and preservation of proteins in the DOC/DON (dissolved organic carbon/nitrogen) pools will elucidate their role in global carbon and nitrogen cycling and will greatly advance our knowledge of marine biogeochemistry.