Shared biomarker development: Common protein sequences across different invertebrates

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  • Published: Sep 1, 2012
  • Author: Steve Down
  • Channels: Proteomics
thumbnail image: Shared biomarker development: Common protein sequences across different invertebrates

Evolutionary conservation

The evolutionary conservation of vitellogenin-like proteins, which are precursors of the major egg yolk protein, allows them to be identified and quantified in unsequenced species that are absent from public databases, say French researchers.

Gammarus fossarum

Image: FERA / DEFRA

The evolutionary conservation of a group of proteins across different species has been examined by a team of scientists based in France who were looking for better ways to identify biomarkers of toxic stress. It is currently very time consuming to study the effects of environmental toxins across many species due to the variability of response across different groups of organisms, or taxa. This variability is partially caused by the molecular diversity between species.

However, some toxins might have similar effects on particular aspects of an organism, such as the reproductive system. In fish, for example, a group of proteins called vitellogenins, which are used to form the major egg yolk protein, are induced by estrogens and have been used as a biomarker of exposure to both natural estrogens and xenoestrogens. Now, vitellogenins are common across many egg-laying organisms, so they might be useful biomarkers in other species too.

The most common method for measuring vitellogenins is enzyme-linked immunosorbent assay (ELISA) but the antibodies used have low inter-species reactivity, so new assays have to be devised for each species. As an alternative, Arnaud Chaumot and colleagues from the National Research Institute of Science and Technology for Environment and Agriculture (Irstea), Lyon, and the University of Lyon decided to look for vitellogenin-like amino acid sequences in different types of invertebrates and to try and exploit them in a mass spectrometric assay.

Vitellogenin sequence search

As a first step, the researchers searched the GenBank and EMBL protein sequence databases to identify vitellogenin-like sequences for a cross section of eight invertebrates that are sensitive to toxins in the wild. They consisted of two bivalves, two insects and one amphipod, isopod, branchiopod and copepod.

The sequences were then used to predict the proteolytic peptides that would be derived from the vitellogenins and the team selected those which would be the strongest for LC-tandem-MS identification. They identified up to five MS/MS transitions that would give the best sensitivity and which conformed to the biological specificity of vitellogenin-like peptides.

The MS/MS transitions were then investigated in practice for proteins extracted from the eight species following peptide formation by digestion with trypsin. The best peptides were further narrowed down by considering those which gave the best biological specificity according to several criteria like the inter-gender ratio.

Protein sequences stretch across different species

The peptides derived from the vitellogenin-like proteins were validated in seven of the eight species, giving rates of peptide sequence confirmation that were typically more than 80%, rising to 100% in some cases. Most of the unconfirmed peptides were due to poor quality fragmentation spectra rather than real false positives.

For the eighth species, which was the insect Tribolium castaneum, all of the candidate peptides that were predicted were actually found in the spectra, but there was poor distinction between the males and females. So, Chaumot proposed that vitellogenin is not involved in the production of egg yolk protein in this species.

This part of the study took six weeks to complete for the seven invertebrates, which is quicker than antibody-based immunoassays would take to develop. The method has been used in another study by the group to quantify vitellogenin in the amphipod Gammarus fossarum where it was used to monitor endocrine disruption in males as well as changes in the reproductive status of females.

In the next step, the researchers used the proteolytic peptides derived from vitellogenins in three species to search for those in some evolutionarily related species, namely four Gammarus species, two Daphnia and two Drosophila. A number of the peptides were found, although the number of matched peptides fell as the species became less related. Nevertheless, the results illustrated the conservation of protein sequence between each taxonomic group.

So, the mass spectrometry technique could be used in ecotoxicological studies of related invertebrate species. The common peptide sequences within the vitellogenin-related proteins will overcome polymorphisms within species and variability across species. This will allow the proteins to be used as biomarkers of the effects of environmental toxins, such as those with estrogenic effects, without having to devise a new method for each species.

Related Links

Environmental Science and Technology 2012, 46, 6515-6523: "Vitellogenin-like proteins among invertebrate species diversity: Potential of proteomic mass spectrometry for biomarker development"

Aquatic Toxicology 2012, 112-113, 72-82: "Vitellogenin-like proteins in the freshwater amphipod Gammarus fossarum (Koch, 1835): Functional characterization throughout reproductive process, potential for use as an indicator of oocyte quality and endocrine disruption biomarker in males"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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