On the use of tandem mass spectra acquired from samples of evolutionarily distant organisms to validate methods for false discovery rate estimation.

Estimating the false discovery rate (FDR) of peptide identifications is a key step in proteomics data analysis, and many methods have been proposed for this purpose. Recently, an entrapment-inspired protocol to validate methods for FDR estimation appeared in articles showcasing new spectral library search tools. That validation approach involves generating incorrect spectral matches by searching spectra from evolutionarily distant organisms (entrapment queries) against the original target search space. Although this approach may appear similar to the solutions using entrapment databases, it represents a distinct conceptual framework whose correctness has not been verified yet. In this viewpoint, we first discussed the background of the entrapment-based validation protocols and then conducted a few simple computational experiments to verify the assumptions behind them. The results reveal that entrapment databases may, in some implementations, be a reasonable choice for validation, while the assumptions underpinning validation protocols based on entrapment queries are likely to be violated in practice. This article also highlights the need for well-designed frameworks for validating FDR estimation methods in proteomics.