MS²Rescore 3.0 Is a Modular, Flexible, and User-Friendly Platform to Boost Peptide Identifications, as Showcased with MS Amanda 3.0

Rescoring of peptide-spectrum matches (PSMs) has emerged as a standard procedure for the analysis of tandem mass spectrometry data. This emphasizes the need for software maintenance and continuous improvement for such algorithms. We introduce MS(2)Rescore 3.0, a versatile, modular, and user-friendly platform designed to increase peptide identifications. Researchers can install MS(2)Rescore across various platforms with minimal effort and benefit from a graphical user interface, a modular Python API, and extensive documentation. To showcase this new version, we connected MS(2)Rescore 3.0 with MS Amanda 3.0, a new release of the well-established search engine, addressing previous limitations on automatic rescoring. Among new features, MS Amanda now contains additional output columns that can be used for rescoring. The full potential of rescoring is best revealed when applied on challenging data sets. We therefore evaluated the performance of these two tools on publicly available single-cell data sets, where the number of PSMs was substantially increased, thereby demonstrating that MS(2)Rescore offers a powerful solution to boost peptide identifications. MS(2)Rescore's modular design and user-friendly interface make data-driven rescoring easily accessible, even for inexperienced users. We therefore expect the MS(2)Rescore to be a valuable tool for the wider proteomics community.