High-Throughput Mass Spectrometry-Based Proteomics with dia-PASEF

Ion mobility separation is becoming an integral part in mass spectrometry-based proteomics. Here we describe the use of a trapped ion mobility-quadrupole time-of-flight (TIMS-QTOF) mass spectrometer for high-throughput label-free quantification with data-independent acquisition. The parallel accumulation-serial fragmentation (PASEF) operation mode positions the mass-selecting quadrupole as a function of the TIMS separation, which allows highly efficient data-independent acquisition schemes (dia-PASEF), but also increases complexity in the method design. We provide a step-by-step protocol for instrument setup, method design, data acquisition and ion mobility-aware, library-based data analysis with Spectronaut. We highlight key acquisition parameters and illustrate their optimization for short gradients. Using the EvosepOne liquid chromatography system, we demonstrate expected results for the analysis of a human cancer cell line at a throughput of 60 samples per day, leading to the quantification of about 6000 protein groups with very high reproducibility. Importantly, the protocol can be readily adapted to other gradients and sample types such as modified peptides.