DIAlignR provides precise retention time alignment across distant runs in DIA and targeted proteomics.

SWATH-MS has been widely used for proteomics analysis given its high-throughput and quantitative reproducibility, but ensuring consistent quantification of analytes across large-scale studies of heterogeneous samples such as human plasma remains challenging. Heterogeneity in large-scale studies can be due to large time intervals between acquisition, acquisition by different operators or instruments or intermittent repair or replacement of parts, such as the liquid chromatography column, all of which affect retention time (RT) reproducibility and successively quantitative performance of SWATH-MS. Here, we present a novel algorithm based on direct alignment of raw MS2 chromatograms using a hybrid dynamic programming approach. The algorithm does not impose a chronological order of elution and allows for aligning of elution-order swapped peaks. Furthermore, allowing RT mapping in a certain window around coarse global fit makes it robust against noise. On a manually validated dataset, this strategy outperforms the current state-of-the-art approaches. In addition, on a real clinical data, our approach outperforms global alignment methods by mapping 98% of peaks compared to 67% cumulatively, is capable of reducing alignment error up to 30-fold for extremely distant runs. The robustness of technical parameters used in this strategy has also been demonstrated. The source code is released under the BSD license at https://github.com/Roestlab/DIAlign.