Fast and Accurate Protein False Discovery Rates on Large-Scale Proteomics Data Sets with Percolator 3.0

TLDR

Percolator is a widely used tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide‑spectrum matches. The study aims to provide reliable protein‑level statistics by comparing protein inference methods and implementing the best‑performing one in Percolator. The chosen method groups proteins by their theoretical peptide sets and considers only the best‑scoring peptide per protein. Percolator 3.0 now processes millions of PSMs in minutes on a commodity computer, as demonstrated by its analysis of 25 million spectra from a draft human proteome study, improving speed over previous limits of hundreds of thousands of PSMs. The source code and binary packages are available from http://percolator.ms/ under an Apache 2.0 license.

Abstract

Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator's processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore, with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method—grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein—in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 million spectra (PM:24870542). The source code and Ubuntu, Windows, MacOS, and Fedora binary packages are available from http://percolator.ms/ under an Apache 2.0 license.