Marine pollution indicators: Comparing protein extractions systems for sentinel species - separationNOW.com

Published/Posted:December 9, 2013- Author: Steve Down - separationNOW.com

Marine ecotoxicology
Since its introduction, proteomics has extended into many different spheres. It is not simply confined to the study of the human condition but extends into other realms including marine toxicology. In this case, when proteomics research is being carried out into the effects of toxic substances on living organisms, it is referred to rather clumsily as marine ecotoxicoproteomics. Despite this cumbersome label, the area is of great importance because it helps to assess damage to the environment that is caused by pollutants.
Any species in a region that is at risk from toxic pollution can be examined by proteomics but there are also some species that are examined more regularly in marine ecotoxicology studies. These sentinel organisms include various members of the mussel family such as the Mediterranean mussel Mytilus galloprovincialis. The creatures are collected in situ and their proteomes are compared with those of control species, to assess the effects of the pollutants.
The first step in this assessment is extraction of the proteins from the tissue. Although this is often regarded as a routine step, it is of critical importance, influencing the protein yields and the detection of low-abundance proteins. Extraction should also be reproducible with minimal technical differences, so that different batches of organisms can be compared confidently.
Three of the most common protein extractions systems have been optimised and compared by a team of Chinese scientists who test them on three sentinel marine organisms. Huifeng Wu and Jianmin Zhao from the Yantai Institute of Coastal Zone Research, with Chenglong Ji and Lei Wei from The Graduate School of Chinese Academy of Sciences, Beijing, reported their findings in Proteomics.

Mussel, flounder and ragworm

The researchers extracted the proteins from the gill tissue of M. galloprovincialis, the liver of the flounder Paralichthys olivaceus and whole bodies of the ragworm Nereis diversicolor. After some initial experimentation with the three solvent systems to improve the quality of the 2D electrophoresis gels of the extracts, their compositions were finalised.
The first system comprised 10% trichloroacetic acid in acetone containing 0.07% dithiothreitol, Tris buffer and enzyme inhibitors. The second was lysis buffer also containing Tris and enzyme inhibitors and the third was the commercial reagent known as TRIzol.
The extracted proteins from the three species were subjected to 2-DE and visualised on the gel by silver staining. Individual proteins were identified following conventional in-gel digestion by mass spectrometric analysis with matching against an animal database.

Different solvent systems for different species

There was no "perfect" extraction system that performed the best for the three organisms. Taking the mussel first, the greatest number of proteins was extracted with TRIzol (1065) followed by TCA-acetone (986) and lysis buffer (920). When the number of matched spots between replicate gels was considered, the order was the same but the numbers were lower at 759, 553 and 505, respectively.
The reproducibility of an extraction system, an important factor in comparative proteomics, was examined with the help of principal components analysis. The PCA plots confirmed that the protein profiles differed according to the extraction protocol but TRIzol was the most reproducible for the mussel. This was in spite of the fact that the other two methods gave higher total protein extraction yields, indicating that yield alone is no criterion for comparison.
With the flounder, lysis buffer extracted far fewer matched proteins than TRIzol and was the poorest of the three systems. The best again proved to be TRIzol, which gave 601 matched proteins compared with 406 with TCA-acetone, and was 19-fold more reproducible. So, TRIzol was the best extractant for mussels and flounder but TCA-acetone was found to be the best for the ragworm with lysis buffer the worst.
Lysis buffer is often preferred for protein recovery because it tends to give higher overall yields but the relatively poor performance shown here suggests that scientists should not automatically turn to it. Apart from producing fewer matched spots on the gels and having lower reproducibility, lysis buffer also extracts relatively few proteins of medium molecular weight, from 25-45 kDa.
This work demonstrates that there is no single ideal system for extracting proteins from marine animals. Based on their results, the researchers recommended TRIzol for extracting proteins from mussel and flounder and TCA-acetone for ragworm when these sentinel organisms are being explored in ecotoxicoproteomics studies.

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