Simulated-to-real Benchmarking of Acquisition Methods in Metabolomics

Data-Dependent and Data-Independent Acquisition modes (DDA and DIA, respectively) are both widely used to acquire MS2 spectra in untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) metabolomics analyses. Despite their wide use, little work has been attempted to systematically compare their performance due to the difficulty and cost of performing comparisons with real experimental data due to the lack of ground truth and the costs involved in running large number of acquisitions. Here, we present a systematic in-silico comparison of these two acquisition methods. To do so, we extended our Virtual Metabolomics Mass Spectrometer (ViMMS) framework with a DIA module. Our results show that the performance of these methods varies with the average number of co-eluting ions as the most important factor. At low numbers, DIA outperforms DDA, but at higher numbers, DDA has an advantage as DIA can no longer deal with the large amount of overlapping ion chromatograms. Results from simulation were further validated on an actual mass spectrometer, demonstrating that using ViMMS we can draw conclusions from simulation that translate well into the real world. Embedding this work within ViMMS also allows for researchers to easily simulate DDA and DIA LC-MS/MS runs, validate them on actual instrument, and potentially prototype novel methods that best combine the characteristics of both approaches. We believe that this work provides a useful guide to the choice of DIA or DDA for scientists involved in metabolomics data acquisition.