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Improved False Discovery Rate Estimation Procedure for Shotgun Proteomics
Interpreting the potentially vast number of hypotheses generated by a shotgun proteomics experiment requires a valid and accurate procedure for assigning statistical confidence estimates to the identified tandem mass spectra. Despite the crucial role such procedures play in most highthroughput proteomics experiments, the scientific literature has not reached a consensus about the best confidence estimation methodology. In this work, we evaluate, using theoretical and empirical analysis, four previously proposed protocols for estimating the false discovery rate (FDR) associated with a set of identified tandem mass spectra: two variants of the target-decoy competition protocol (TDC) of Elias and Gygi and two variants of the separate target-decoy search protocol of Kall et al. Our analysis reveals signi ficant biases in the two separate target-decoy search protocols. Moreover, the one of the TDC protocol that provides an unbiased estimate FDR among the target PSMs does so at the cost of forfeiting a random subset of high-scoring spectrum identifications. We therefore propose the mix-max procedure to provide unbiased, accurate FDR estimates in the presence of a well-calibrated scores. The method avoids biases associated with the two separate target-decoy search protocols and also avoids the propensity for target-decoy competition to discard a random subset of high-scoring target identifications