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

Animal testing has long been the cornerstone of chemical safety assessments, but fish embryo assays represent an alter­native. Omics studies allow the examination of early molecular responses of organisms to environmental stressors, but reduction of animal use within this context has been overlooked. For proteomics, there is significant disparity and vari­ability in the organismal pool size used for studies, ranging from 1-1500 embryos per replicate for zebrafish alone. However, it is unknown if varying sample pool size results in differences in protein identifications. To examine whether the detected proteome changes depend on this variable, 3 pool sizes (5, 10 or 20 embryos or larvae per replicate) were compared using the two most common fish models with an appropriate biological replicate number determined by power analysis (n = 7). Data was acquired using MSe, resulting in 1,946 and 3,172 protein groups identified (1% false discovery rate) for fathead minnow and zebrafish, respectively. Proteins were not differentially expressed among pool sizes, and no significant difference was observed among the identified protein groups. However, for the fathead minnow, a decrease in the number of identified proteins was observed with increasing pool size, while a trend towards an increase in protein identifications was observed in zebrafish between the lowest and highest pool size. Taken together, our observations suggest that a proteome characterization experiment using these fish models can achieve comparable protein identifications using pool sizes of less than 5 organisms per replicate, assuming a protein requirement of 50 μg or less.