Bekker-Jensen DB, Bernhardt OM, Hogrebe A, Martinez-Val A, Verbeke L, Gandhi T, Kelstrup CD, Reiter L, Olsen JV. Nature Communications. Protein phosphorylation is a predominant post-translational modification that dynamically regulates protein function and cell signaling. In this collaboration with the Olsen group at the University of Copenhagen, we showed that DIA allows reliable phosphosite localization for hundreds of samples and outperforms DDA in dynamic range, reproducibility, sensitivity, and accuracy. Additionally, directDIA, the library-free workflow of Spectronaut reaches phosphoproteome depths comparable to library-based DIA analysis.