TIMS ² Rescore: A DDA-PASEF optimized data-driven rescoring pipeline based on MS ² Rescore

Abstract The high throughput analysis of proteins with mass spectrometry (MS) is highly valuable for understanding human biology, discovering disease biomarkers, identifying therapeutic targets, and exploring pathogen interactions. To achieve these goals, specialized proteomics subfields – such as plasma proteomics, immunopeptidomics, and metaproteomics – must tackle specific analytical challenges, such as an increased identification ambiguity compared to routine proteomics experiments. Technical advancements in MS instrumentation can counter these issues by acquiring more discerning information at higher sensitivity levels, as is exemplified by the incorporation of ion mobility and parallel accumulation - serial fragmentation (PASEF) technologies in timsTOF instruments. In addition, AI-based bioinformatics solutions can help overcome ambiguity issues by integrating more data into the identification workflow. Here, we introduce TIMS 2 Rescore, a data-driven rescoring workflow optimized for DDA-PASEF data from timsTOF instruments. This platform includes new timsTOF MS 2 PIP spectrum prediction models and IM2Deep, a new deep learning-based peptide ion mobility predictor. Furthermore, to fully streamline data throughput, TIMS 2 Rescore directly accepts Bruker raw mass spectrometry data, and search results from ProteoScape and many other search engines, including MS Amanda and PEAKS. We showcase TIMS 2 Rescore performance on plasma proteomics, immunopeptidomics (HLA class I and II), and metaproteomics data sets. TIMS 2 Rescore is open-source and freely available at https://github.com/compomics/tims2rescore .