Award: OCE-1233014

It is well known that proteins are a major contributor to organic carbon and nitrogen in the living ocean. Further, a large body of evidence assembled over several decades has documented that their amino acid building blocks comprise an important fraction (typically 40-60%) of the total nitrogen identified in coastal and oceanic waters and preserved in sediments. This foundational research established the importance of proteins as both a major fraction of organic matter and as a participant in organic matter recycling. Over the last decade, tandem mass spectrometry (MS/MS) methods developed for the analysis of peptides and protein reconstruction have been adapted from the biomedical community for environmental proteomics approaches (e.g., see www.environmentalproteomics.org). Here, we set out to identify protein(s) synthesized by heterotrophic marine bacteria during initial stages of protein degradation as a means of identifying their contribution to these processes in the ocean. Additionally, we identify the major biochemical pathways operative during protein degradation. The goal of this research was to provide us with a basic understanding of which proteins would increase in abundance during degradation so that we could target these proteins for downstream analyses. Additionally, we determined the limits of detection in mixed community proteomic samples using several different mass spectrometry acquisition strategies. We designed an experiment to follow the uptake of the only carbon food source (13C6-leucine) by Ruegeria pomeroyi using proteomics. The bacteria, R. pomeroyi, was grown in culture and maintained as a control group or given a 13C6-labeled leucine substrate. Samples from the control and experimental groups, in total, were examined via mass spectrometry in order to identify and quantify total and labeled proteins produced through time. Although hundreds of proteins were labeled within hours of the 13C6-leucine addition, the most abundantly labeled proteins were those one might expect to rise with cellular division and energy production. However, the unexpected, intriguing finding was that much of the 13C6-leucine was not degraded within the cell once it was ingested but instead was used directly in many proteins unaltered. Further, branched-chain-amino-acid ABC transporters increased in abundance and were labeled with 13C6-leucine demonstrating that the R.pomeroyi produces specific transporters responsible for acquiring the needed substrate using the very substrate provided. This suggests that we could monitor degradation of a natural bloom by tracking the transporters expressed by the specific bacteria present to understand who is doing what during the degradation process. Through a collaboration with the Junge lab (UW) we explored how a prominent psychrophilic bacteria in the Arctic and Antarctic survive subzero temperatures over winter. Colwellia psychrerythraea was cultured at -1°C, -5°C, -10°C, and -20°C and tritiated thymidine and leucine were monitored to track DNA and protein synthesis over an 8 weeks. In order to identify potential molecular strategies that enable 34H cells to maintain cellular integrity under sub-zero temperatures in hyper-saline environments, we examined proteomic profiles of 34H after 8 weeks of -10°C exposure (Figure 1). We identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo-taxis and sensing (Figure 2). By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold-adapted marine organisms to sustain cellular function in their habitat, having implications on cryogenics and cryopreservation (Nunn et al., 2015). Another of our primary objectives was to determine the detection limit of specific bacterial peptides when mixed with eukaryotes as the dominant matrix. This objective was added so we could optimize our mass spectrometry methods to improve our ability to detect active bacteria living on particulate organic matter. This was completed using a dilution curve to understand what our analytical capabilities are when using mass spectrometry to target a desired peptide when mixed with a complex matrix of hundreds of thousands of undesired peptides (Figure 3). We were able to target and detect bacterial peptides down to a ratio of 5:1 bacterial to phytoplankton protein (Figure 4). This ratio is similar to what is observed when looking at dead phytoplankton biomass that is actively degraded by bacteria. The research demonstrated that standard methods using data dependent acquisition mass spectrometry cannot detect these bacterial peptides, but targeted SRM mass spectrometry can (Timmins-Shiffman et al., in review). This funding has allowed us to establish fundamental methods for detecting newly synthesized proteins by active bacteria living on dead biomass present in the ocean to better understand who is doing what during the degradation process. These tools will allow us to better track how mixed microbial communities control the carbon and nitrogen cycles in our changing environment (May et al., 2016; Timmins-Schiffman et al., 2016). Last Modified: 10/06/2016 Submitted by: Brook L Nunn