Electrophoretic Extraction and Proteomic Characterization of Proteins Buried in Marine Sediments

Eli Moore,David Goodlett,Jessica Faux,H Harvey,Brook Nunn

doi:10.3390/chromatography1040176

Abstract

Proteins are the largest defined molecular component of marine organic nitrogen, and hydrolysable amino acids, the building blocks of proteins, are important components of particulate nitrogen in marine sediments. In oceanic systems, the largest contributors are phytoplankton proteins, which have been tracked from newly produced bloom material through the water column to surface sediments in the Bering Sea, but it is not known if proteins buried deeper in sediment systems can be identified with confidence. Electrophoretic gel protein extraction methods followed by proteomic mass spectrometry and database searching were used as the methodology to identify buried phytoplankton proteins in sediments from the 8–10 cm section of a Bering Sea sediment core. More peptides and proteins were identified using an SDS-PAGE tube gel than a standard 1D flat gel or digesting the sediment directly with trypsin. The majority of proteins identified correlated to the marine diatom, Thalassiosira pseudonana, rather than bacterial protein sequences, indicating an algal source not only dominates the input, but also the preserved protein fraction. Abundant RuBisCO and fucoxanthin chlorophyll a/c binding proteins were identified, supporting algal sources of these proteins and reinforcing the proposed mechanisms that might protect proteins for long time periods. Some preserved peptides were identified in unexpected gel molecular weight ranges, indicating that some structural changes or charge alteration influenced the mobility of these products during electrophoresis isolation. Identifying buried photosystem proteins suggests that algal particulate matter is a significant fraction of the preserved organic carbon and nitrogen pools in marine sediments.

Highlights

Much of the ocean is influenced by nitrogen limitation [1], and understanding marine protein cycling is important for tracking the global organic nitrogen cycle
The majority of identified peptides and proteins correlated to T. pseudonana chloroplast proteins, including the RuBisCO large subunit and three fucoxanthin chlorophyll a/c binding proteins (FCPs)
The denaturing conditions of the SDS-PAGE gels used in this study indicate that charge alteration could play a role in protein mobility

Summary

Introduction

Much of the ocean is influenced by nitrogen limitation [1], and understanding marine protein cycling is important for tracking the global organic nitrogen cycle. Solid state NMR has provided evidence that the majority of organic nitrogen in dissolved and particulate marine organic matter contains amide bonds as found in proteins [2,3,4]. Protein building blocks, such as total hydrolysable amino acids (THAAs, total amino acids that can be extracted using 6 N HCl), are found to account for up to 30%–40%. Identifying intact proteins and peptides buried in marine sediments would give valuable information towards understanding marine biogeochemical cycles and reconstructing algal/microbial populations [9,10]. New bioseparation methods are needed to successfully extract proteins from deep sediments for subsequent analyses

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Results

Conclusion