Resolving proteins in seawater

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  • Published: May 1, 2005
  • Author: Steve Down
  • Channels: Proteomics & Genomics / Proteomics
thumbnail image: Resolving proteins in seawater

Seas and oceans contain huge stores of dissolved organic matter but much of it remains uncharacterised. The dissolved protein content in the Gulf of Mexico has been examined in a rare marine proteomics study.

Dissolved organic matter (DOM), also known as dissolved organic carbon (DOC) is a general term used to describe the many thousands of organic compounds in water that originate from organic materials. They are derived from plants or animals and being dissolved, they are distinct from the particulate matter that is also found. DOM represents one of the largest reservoirs of carbon in the world, accounting for about 700 x 1015 g of carbon, compared with the estimated 750 x 1015 g of carbon contained in atmospheric carbon dioxide and the annual flow of 7 x 1015 g of carbon from anthropogenic activities.

The oceans are an important medium for maintaining the global carbon dioxide balance, absorbing about 33% of that produced by tropical deforestation and burning fossil fuels. But they have also been collecting organic carbon for thousands of years, from marine species, from the air, and from matter swept off the land. In the oceans, DOM has been classified into two distinct types of matter. The majority (>99%) is constantly changing at a fast turnover rate and represents a nutritional source for oceanic microorganisms.

The minor DOM species (<1%) are known as the refractory fraction and comprise stable compounds, mostly marine-derived polymeric materials, that are up to 6000 years old, or older. Although much of this fraction remains uncharacterised, it is known that proteins make up a significant proportion. There have been few large-scale studies to identify marine proteins due to their complex structures. Now, US researchers have conducted a proteomics study of dissolved proteins in seawater using modern proteomics technology.

Aaron Timperman and colleagues from the Department of Chemistry at West Virginia State University with Carlos Del Castillo from the Stennis Space Centre (NASA) took samples from the Gulf of Mexico. The dilute nature of the seawater meant that large volumes needed to be processed to obtain sufficient proteins to work with. So 60 L of seawater was concentrated using tangential flow ultrafiltration followed by centrifugal ultrafiltration to give 10 mL of sample. The proteins present were precipitated and the pellet was analyzed in two ways, as described in Marine Chem. 2005.

Firstly, the whole pellet was digested with trypsin and the peptide mixture was analyzed by LC/MS/MS and LC/LC/MS/MS with electrospray ionisation. Alternatively, the proteins in the pellet were initially fractionated by SDS-PAGE before in-gel digestion of the individual protein spots and LC/MS/MS analysis. The MS/MS spectra obtained in both cases were searched against DNA and protein databases and were also subjected to de novo peptide sequencing. The latter technique generated peptide sequence tags that were used to search protein databases for protein classes.

Less than 25 proteins were detected by gel electrophoresis, confirming that a small number of refractory proteins exist in seawater at detectable concentrations. Their molecular masses ranged from 20,000 to more than 100,000 Da. A low-level background was also detected, probably containing more dilute proteins, humic substances and degradation products.

The protein similarity searches fitted some of the proteins into two classes: membrane/envelope proteins and enzymes, but the majority of proteins were non-classifiable because the peptide tag sequence was found across several protein and organism classes. Even so, the categorization revealed for the first time that there are other proteins present in seawater apart from bacterial membrane proteins. The enzymes long-chain fatty acyl synthetase, anthranilate synthase and ribulose bisphosphate carboxylase were identified.

The results also confirm that two modes of protein preservation are mutually involved. In physical protection, the proteins are implanted on or trapped within membrane fragments, so that they are shielded from attack. In selective preservation, the folded protein structure provides inherent protection against degradation.

Future work will focus on multiple enzyme digests to give longer peptide sequences that will permit modelling studies of secondary structure. These, in turn, will help to establish the survival mechanisms of marine proteins and clarify the role of DOM in global carbon cycling involving carbon dioxide and other greenhouse gases.


The murky Mississippi flowing into the Gulf of Mexico (Image: courtesy Liam Gumley, Space Science and Engineering Center, University of Wisconsin-Madison and the MODIS science team)

DOM in the oceans is at the bottom of the food chain

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