The proteomic study of Danio rerio embryos using one-dimensional electrophoresis and mass-spectrometry

Abstract

Using one dimensional proteomic mapping (combination of one dimensional gel electrophore sis (1DE) with subsequent mass spectrometry MALDI TOF PMF) the protein profile of Danio rerio embryos has been investigated. The fish species Danio rerio is the most effective alternative model of verte brates used for studies of drug toxicity (e.g. doxorubicin) due to its high degree of homology with human genome. The proteomic profiling resulted in identification of 84 proteins, including 15 vitellogenins. Using the procedure of preparation of homogenates of Danio rerio embryos optimized by ultrasonic treatment pro moting removal of yolk basic proteins (vitellogenin) we have registered changes in the proteome profile of D. rerio embryos induced by doxorubicin (DOX). Growth D. rerio embryos in the medium with DOX caused the decrease in the number of vitellogenins, disappearance of cardiac troponins, and induction of caspase 3. All these observations are consistent with the literature data on doxorubicin induced cardiotoxicity. The pro posed method of 1D proteomic mapping may be used not only for protein identification but also for registra tion of changes in embryonic proteomic profile caused by drugs or any toxic compound for studying the mechanisms underlying induced toxicity.

One-dimensional proteomic profiling of Danio rerio embryo vitellogenin to estimate quantum dot toxicity

“…In this study, the sequential thin slicing of an SDS-PAGE gel and subsequent slice-by-slice MALDI-TOF MS were used to comprehensively analyze the Vtg cleavage products from D. rerio embryos. This proteomic methodology has previously been used to distinguish the polymorphic variants of human liver cytochromes P450 [ 32 , 33 ] and to study doxorubicin toxicity in a D. rerio embryo model [ 34 ].…”

“…We used PMF to identify the D. rerio embryonic proteins for each mass spectrum, across all the samples, slices, and replicates, using a previously reported strategy for obtaining and processing thin gel slices in selected regions of the SDS-PAGE gel [ 33 ]. The resulting slices did not usually correspond to protein bands and contained different amounts of the total protein, which was visible as irregular Coomassie staining within the gel regions (Figure 1 b).…”

“…Therefore, for in-gel digestion, we calculated the amount of trypsin per gel slice. This equation was previously established and validated using human liver microsomal proteins [ 33 ] and embryonic D. rerio proteins after doxorubicin exposure [ 34 ].…”

“…We previously proposed a one-dimensional proteomic mapping approach based on the cutting of SDS-PAGE gel lanes and the subsequent MALDI-TOF peptide mass fingerprinting (PMF) analysis of each slice, to increase the number of proteins identified [ 32 , 33 ]. We evaluated the application of this approach to the study of the D. rerio protein profile and its changes in response to doxorubicin exposure [ 34 ].…”

Proteomic analysis of ametryn toxicity in zebrafish embryos

“…Comparatively, SDS‐PAGE displays high reproducibility and broad resolution, allowing the separation of proteins with apparent MWs of 10–450 kDa . Kisrieva et al proposed a one‐dimensional proteomic mapping approach based on the cutting of SDS‐PAGE gel lanes and the subsequent MALDI‐TOF peptide mass fingerprinting (PMF) analysis of each slice, to increase the number of proteins identified. Here, we utilized the same strategy to analyze the one‐dimensional proteomic profiles of zebrafish embryos in response to exposure to AMT.…”