Optimal replication and the importance of experimental design for gel-based quantitative proteomics.

Quantitative proteomic studies, based on two-dimensional gel electrophoresis, are commonly used to find proteins that are differentially expressed between samples or groups of samples. These proteins are of interest as potential diagnostic or prognostic biomarkers, or as proteins associated with a trait. The complexity of proteomic data poses many challenges, so while experiments may reveal proteins that are differentially expressed, these are often not significant when subjected to rigorous statistical analysis. However, this can be addressed through appropriate experimental design. A good experimental design considers the impact of different sources of variation, both analytical and biological, on the statistical importance of the results. The design should address the number of samples that must be analyzed and the number of replicate gels per sample, in the context of a particular minimum difference that one is seeking to achieve. In this study, we explore the ways to improve the quality of protein expression data from 2-DE gels, and describe an approach for defining the number of samples required and the number of gels per sample. It has been developed for the simplest of situations, two groups of samples with variation at two levels: between samples and between gels. This approach will also be useful as a guide for more complex designs involving more than two groups of samples. We describe some Internet-accessible tools that can assist in the design of proteomic studies.