Data provided report the use of trapped ion mobility spectrometry (TIMS) to fractionate ions in the gas phase based on their ion mobility (V⋅s/cm2) followed by parallel accumulation serial fragmentation (PASEF) in a quadrupole time-of-flight instrument. TIMS fractionation coupled to DDA-PASEF allowed for the detection of approximately 7,000 proteins and over 70,000 peptides per overall run from 200ng of human (HeLa) cell lysate per injection using a commercial UPLC column with a 90-minute gradient. This project also explored the utility of TIMS fractionation to generate a DDA library for downstream DIA analysis using shorter LC gradients (20 minutes) as well as lower sample input. Using a 20min gradient, we identified 4,092 and 6,654 proteins on average per run, respectively, from 10ng and 200ng of human (HeLa) cell lysate input based on a TIMS-fractionated library consisting of 82,214 peptides derived from 7,615 proteins.