Scoring information integration with statistical quality control enhanced cross-run analysis of data-independent...

The peptide-centric strategy is widely applied in data-independent acquisition (DIA) proteomics to analyze multiplexed MS2 spectra. However, current software tools often rely on single-run data for peptide peak identification, leading to inconsistent quantification across heterogeneous datasets. Match-between-runs (MBR) algorithms address this by aligning peaks or elution profiles post-analysis, but they are often ad hoc and lack statistical frameworks for controlling peak quality, causing false positives and reduced quantitative reproducibility. Here we present DreamDIAlignR, a cross-run peptide-centric tool that integrates peptide elution behavior across runs with a deep learning peak identifier and alignment algorithm for consistent peak picking and FDR-controlled scoring. DreamDIAlignR outperformed state-of-the-art MBR methods, identifying up to 21.2% more quantitatively changing proteins in a benchmark dataset and 36.6% more in a cancer dataset. Additionally, DreamDIAlignR establishes an improved methodology for performing MBR compatible with existing DIA analysis tools, thereby enhancing the overall quality of DIA analysis.