44963-AC2
Bacterial Proteins in Sediments: Implications for Sources and Cycling of Ancient Organic Matter
Despite the fact that proteins represent the major biosynthetic source for organic nitrogen in the biosphere, its potential to contribute to pool of uncharacterized nitrogen in sediments and nitrogenous compounds observed in petroleum remains elusive. Detailed analysis of the material that remains preserved in the geological record reveals a complex mixture of organic molecules, typically as mixtures of a few individual molecules and the majority as undefined, macromolecular material often referred to as humic substances. The primary aim of this project is to investigate the possibility that protein is an important starting material for the organic nitrogen ultimately preserved in ancient sediments and to begin to identify its sources. As major recyclers in the environment, bacteria represent an unknown contributor to much of the organic matrix, and we hypothesize their varied organic composition have allow their importance to be underestimated. Recent advances in peptide mapping now allow us to examine the primary sequence of proteins in complex mixtures and provide the potential to examine complex suites of products preserved in organic matrices. The goal of this project is to provide a more quantitative picture on the origin of proteins which might be preserved and the potential contribution of bacterial sources to sedimentary organic nitrogen.
Over the first year much effort went into developing protocols which could maximize the extraction of proteins or their products from the complex matrices of sediments with minimal alteration. Standard biochemical methods do not allow for the highly heterogeneous matrix of sediments to be easily overcome and a range of approaches were examined to adapt biochemical methods to environmental applications. These included the evaluation and adaptation of standard extraction protocols used on biological matrices, revised cleanup approaches using one dimensional electrophoresis and the development of new approaches including solid phase sorption followed by organic solvent elution to recover extractable proteins products. In addition to extraction and recovery, a parallel effort included experiments to investigate the hydrolysis and recovery of peptides from a model protein which has recognizable sequence coverage from multiple peptide subunits. Cytochrome C was chosen for high fraction (80%) of recognizable peptides and introduced into the sediment matrix to better understand the possible interferences to recovery of both intact proteins and the hydrolysed products that also exist. Using proteomics based mass spectrometry; we followed its hydrolysis and recovery from sediments. These experiments have shown that protein hydrolysis is rapid, with a large portion of the introduced protein rapidly recycled. Specific hydrolysis points for Cytochrome C were observed, however, with a number of peptides associated with the heme core of the protein retained throughout the incubation process. These results suggest that bacterial proteins specific to reaction centers (e.g. glycated proteins and binding proteins with metal cofactors) are possible targets for preservation and recovery.
Building on these results, over the last year we have begun to examine detrital material and sediments to extract protein or their modified fragments from complex matrices. The investigation of extraction protocols has shown high variability depending on the rigor of the extraction. Using total amino acid content of materials as a metric for recovery, we have found that biochemical approaches (gentle buffers) do not proved quantitative recoveries, but contain a greater fraction of more intact proteins (as high molecular weight material) verses harsh methods such as NaOH. The approach chosen is thus a balance between recovery efficiency and preservation; it relies on a buffer base with the addition of surfactants and solubilisation reagents to maximize recovery of intact material which contain greater information potential. The doctoral student supported by this project (Eli Moore) presented results of the analytical challenges at the 2008 Ocean Sciences meeting in an oral presentation. We also presented results of our new collaboration with proteomics specialists (Dr. Brook Nunn, Univ. Washington Dept of Medicinal Chemistry) who is assisting with the database development needed to decipher peptide fragment information and related products in complex matrices. In the final phase of this project we will be applying these approaches to field collected material, including recent and ancient sediments. The use of optimized extraction approaches and our potential target proteins are now being examined in Bering Sea and sedimentary environments in the hopes of recovering protein products and their history of organic matter deposition.
