High-Coverage Four-Dimensional Data-Independent Acquisition Proteomics and Phosphoproteomics Enabled by Deep Learning-Driven Multidimensional Predictions

Four-dimensional (4D) data-independent acquisition (DIA)-based proteomics is a promising technology. However, its full performance is restricted by the time-consuming building and limited coverage of a project-specific experimental library. Herein, we developed a versatile multifunctional deep learning model Deep4D based on self-attention that could predict the collisional cross section, retention time, fragment ion intensity, and charge state with high accuracies for both the unmodified and phosphorylated peptides and thus established the complete workflows for high-coverage 4D DIA proteomics and phosphoproteomics based on multidimensional predictions. A 4D predicted library containing ∼2 million peptides was established that could realize experimental library-free DIA analysis, and 33% more proteins were identified than using an experimental library of single-shot measurement in the example of HeLa cells. These results show the great values of the convenient high-coverage 4D DIA proteomics methods.