High sample throughput in proteomics, like that used in genomics, is highly desirable. Moreover, the highest analytical depth in proteomics is only achieved on fractionated samples, requiring subsequent analysis with short gradients to achieve reasonable overall measurement times per sample. The timsTOF Pro with trapped ion mobility spectrometry (TIMS) offers additional separation power and increased peak capacity. The powerful Parallel Accumulation Serial Fragmentation (PASEF) method (Meier et al., JPR 2015) for very high sequencing speed is perfectly suited for proteome analysis on short gradients. We have optimized MS conditions, column lengths and LC overhead times to obtain runs of 28.8 min injection to injection (50 samples/day) on the nanoElute (Bruker Daltonik) and demonstrate applicability for high throughput application.