Ion mobility‐resolved phosphoproteomics with dia‐PASEF and short gradients

Mass spectrometry‐based phosphoproteomics has identified >150,000 post‐translational phosphorylation sites in the human proteome. To disentangle their functional relevance, complex experimental designs that require increased throughput are now coming into focus. Here, we apply dia‐PASEF on a trapped ion mobility (TIMS) mass spectrometer to analyze the phosphoproteome of a human cancer cell line in short liquid chromatography gradients. At low sample amounts equivalent to ∼20 ug protein digest per analysis, we quantified over 13,000 phosphopeptides including ∼8700 class I phosphosites in 1 h without a spectral library. Decreasing the gradient time to 15 min yielded virtually identical coverage of the phosphoproteome, and with 7 min gradients we still quantified about 80% of the class I sites with a median coefficient of variation