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44963-AC2
Bacterial Proteins in Sediments: Implications for Sources and Cycling of Ancient Organic Matter

H. Rodger Harvey, University of Maryland Center for Environmental Science

Despite the fact that proteins represent the major source for organic nitrogen in the biosphere, its potential to contribute to the organic nitrogen of sediments and the actual sources of protein or its products 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. Given that bacteria represent the major recyclers in the environment, remnant microbial proteins are hypothesized as likely contributors to the sedimentary nitrogen record and the specific target for our analytical efforts. Recent advances in peptide mapping now allow us to examine the primary sequence of proteins in complex mixtures and the broad suite of products preserved in organic matrices. The goal of this project is to integrate these approaches to determine the origin of proteins which might be preserved and the potential for bacteria to be major contributors to sedimentary organic nitrogen.

Over the first year of this project much effort has gone into developing protocols which can 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 have been examined to adapt biochemical methods to environmental applications. These have included evaluation and adaptation of standard extraction protocols used for 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 products. In addition to extraction and recovery, a parallel effort has 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 have followed its hydrolysis and recovery in sediments and the impact of the native bacterial assemblage on the rates and specificity of peptide loss and recovery (Squier and Harvey, 2007). These experiments have shown that protein hydrolysis is rapid in sediments 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. The use of optimized extraction approaches and potential target proteins are now being applied to sediments from several ocean basins and environments for analysis by tandem mass spectrometry in the hopes of recovering protein products preserved.

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