Protein responses in blue mussels (Mytilus edulis) exposed to organic pollutants: a combined CYP-antibody/proteomic approach

Polyclonal antibodies were raised against highly conserved, trans-metazoan sequences of cytochrome P450 (CYP) families 2 and 4 and used to investigate responses in the common blue mussel (Mytilus edulis) exposed to various organic contaminants. The results were evaluated by means of cross-reacting proteins on Western blots of both one- and two-dimensional electrophoresis gels, and by scanning spectroscopy measurements of total CYP content. Furthermore, a proteomic approach was applied aimed at elucidating exposure-related protein changes in a more general term. Identities of isolated proteins were searched by means of peptide mass fingerprints obtained from MALDI-TOF MS analyses. The results demonstrated that both antibodies rendered several cross-reactive bands when probed on Western blots. The most obvious cross-reaction of the CYP2 antibody was with a strongly expressed protein of size approximately 57kDa, pI 4.5-4.6, whereas the CYP4 antibody cross-reacted with a protein of size approximately 55kDa, pI 5.6. However, expression of cross-reacting proteins did not change as a result of the exposures, and resulted only in small and insignificant fluctuations in total CYP content. As a contrast, silver-stained 2DE gels showed that several microsomal proteins were affected in individuals exposed to diallylphthalate as well as crude oil, with and without a spike of alkylphenols and PAHs. Mass spectrometry based analyses of excised, trypsin-digested spots did so far not decipher the identities of the proteins affected by the exposures, nor of those cross-reacting with CYP2 and CYP4 antibodies. This study has underlined the power of the proteomic approach in environmental toxicology, although protein identification was not successful. The missing identities of the proteins cross-reacting with the CYP2- and CYP4-antibodies does not enable a clear conclusion as to whether or not these peptides actually represent CYP iso-enzymes.