Proteomics-Based Method for the Assessment of Marine Pollution Using Liquid Chromatography Coupled with Two-Dimensional Electrophoresis

Abstract

Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel "blue mussel" (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS), and they are involved in beta-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.

Review of recent proteomic applications in aquatic toxicology

“…Using protein expression profiles, several authors have reported on the use of proteomic approaches for the establishment of ''protein signatures'' after exposure to a variety of environmental contaminants including PCBs, polyaromatic hydrocarbons (PAHs), heavy metals [50,51], and crude oil [52][53][54]. Additionally, proteomics has been used to compare profiles of mussels residing in polluted areas to those of reference sites [55,56]. Not only have the proteomes of these bivalve species been evaluated when exposed to individual contaminants, but to mixtures as well [57].…”

“…Conversely, upwards of 39 C. riparius larval proteins were identified through MALDI-TOF-MS and spectra blasted against the National Center for Biotechnology Information nonredundant protein database, or Mass Spectrometry protein sequence database (MSDB) with MS-Fit program [67]. However, many proteomic studies with aquatic invertebrates have identified, on average, 15 proteins [8,43,56,66], indicating the problem with matching amino acid sequences across species. Some studies have tried various techniques to improve protein identification by incorporating fractionation of samples, as well as the use of nanospray-ion trap MS, followed by database searching and de novo sequencing [3].…”

Effects of pesticides and antibiotics on penaeid shrimp with special emphases on behavioral and biomarker responses

“…Some authors have reported that environmental stress such as variation in salinity and temperature [96] or exposure to environmental contaminants such as heavy metals, xenoestrogen, and chlorinated compounds [94,97,98] has an impact on protein expression in different tissues of relevant aquatic organisms. Although gel-free methods using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry have been investigated [99][100][101], environmental biomarker discovery still relies mainly on 2D gel electrophoresis and, to our knowledge, is lacking for aquaculture production [102][103][104]. However, there is no doubt that using a proteomic approach in combination with other biomarkers at physiological, behavioral, and biochemical levels will give a more precise set of biomarkers for assessing the exposure of penaeid shrimp to drugs and chemicals.…”

Proteomic Analysis of Mussels Exposed to Fresh and Weathered Prestige’s Oil

“…Digestive glands were processed following a protocol for sample prefractionation and 2-DE protein separation already described (Amelina et al, 2007). Briefly, digestive glands were homogenized with the aid of a pestle and AG®501-X8 Resin glass beads (BioRad Laboratories, Inc., Hercules, CA, USA) in a homogenization buffer containing a protease inhibitor cocktail.…”

“…Precipitated proteins were solubilized in a solubilization buffer described by Rabilloud with some modifications (Rabilloud, 1998;Amelina et al, 2007), alkylated with 30 mM iodoacetamide (IAA) in darkness and mixed with a rehydration buffer previous to the isoelectrofocusing (IEF) step. Proteins (300 µg) were loaded in the PROTEAN® IEF Cell (BioRad Laboratories) tray and IPG strips (11 cm, pH range of 4-7, BioRad Laboratories) placed on top.…”