Exploratory analysis of the application of animal reduction approaches in proteomics: How much is enough?_suppl

Abstract

Animal testing has been the corner stone for chemical safety assessments, but fish embryo assays now represent an alternative. Increases in omics studies offers unparalleled access to examine early molecular responses in organisms in response to environmental stressors and yet reduction in animal usage within this context has been overlooked. For proteomics, there is significant disparity and variability in organismal pool size ranging from 1-2000 embryos per replicate for zebrafish alone. However, it is unknown if varying sample pool size results in higher protein identifications. To examine whether the proteome changes are dependent on this variable, 3 pool sizes (5, 10 or 20 embryos per replicate) were examined using the two most common fish models with appropriate biological replicate number determined based on statistical power analysis (n=7). Samples were analysed by data-independent acquisition (DIA), resulted in 1,946 and 2,770 protein groups identified (1 % FDR) for the fathead minnow and zebrafish, respectively. Proteins were not differentially expressed among pool sizes, and no significant difference was observed among the protein groups identified. However, for the fathead minnow, a decrease in the number of proteins identified was observed with increasing pool size, while only a modest increase of 110 protein identifications was observed in zebrafish between the lowest and highest pool size. Taken together, our observations suggests that a proteome characterization experiment using these fish models can achieve comparable protein identifications using pool sizes of less than 5 per replicate assuming a protein requirement of 50µg or less.