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 here introduce MS²Rescore 3.0, a versatile, modular, and user-friendly platform designed to increase peptide identifications. Researchers can install MS²Rescore across various platforms with minimal effort and benefit from a graphical user interface, a modular Python API, and extensive documentation. To showcase benefits of this new version, we connected MS²Rescore 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 MS²Rescore offers a powerful solution to boost peptide identifications. The modular design and user-friendly interface make MS²Rescore a valuable tool for the proteomics community, offering additional insights into the often nontransparent topic of machine learning, making it more accessible even for inexperienced users.