Extensive and accurate benchmarking of DIA acquisition methods and software tools using a complex proteomic standard

In the past few years, LC-MS/MS in DIA mode has become a strategy of choice for deep coverage of complex proteomes and accurate quantification of low abundant species. However, there is still no consensus in the literature on the best acquisition parameters and processing tools to use. We present here the largest benchmark of DIA proteomic workflows on Orbitrap instruments ever published. Using a complex proteomic standard, we tested 36 workflows including 4 different acquisition schemes and 6 different software tools (DIA-NN, DIA-Umpire, OpenSWATH, Scaffold-DIA, Skyline and Spectronaut-Pulsar-X) with or without the use of a spectral library. By reporting the protein identifications, linearity, reproducibility, as well as sensitivity and specificity in 28 pairwise comparisons, we propose here a guide book for the users to choose the best DIA strategy for their data. Moreover, our data has been deposited on ProteomeXchange for testing or developing new DIA processing tools.