Method for Independent Estimation of the False Localization Rate for Phosphoproteomics

Phosphoproteomic methods are commonly employed to identify and quantify phosphorylation sites on proteins. In recent years, various tools have been developed, incorporating scores or statistics related to whether a given phosphosite has been correctly identified or to estimate the global false localization rate (FLR) within a given data set for all sites reported. These scores have generally been calibrated using synthetic datasets, and their statistical reliability on real datasets is largely unknown, potentially leading to studies reporting incorrectly localized phosphosites, due to inadequate statistical control. In this work, we develop the concept of scoring modifications on a decoy amino acid, that is, one that cannot be modified, to allow for independent estimation of global FLR. We test a variety of amino acids, on both synthetic and real data sets, demonstrating that the selection can make a substantial difference to the estimated global FLR. We conclude that while several different amino acids might be appropriate, the most reliable FLR results were achieved using alanine and leucine as decoys. We propose the use of a decoy amino acid to control false reporting in the literature and in public databases that re-distribute the data. Data are available via ProteomeXchange with identifier PXD028840. 2022 The Authors.