Speedy-PASEF: Analytical flow rate chromatography and trapped ion mobility for deep high-throughput proteomics

Increased throughput in proteomic experiments can improve accessibility of proteomic platforms, reduce costs and facilitate new approaches in systems biology and biomedical research. Here we propose Speedy-PASEF, a combination of analytical flow rate chromatography with ion mobility separation of peptide ions, data-independent acquisition and data analysis with the DIA-NN software suite, for conducting fast, high-quality proteomic experiments that require only moderate sample amounts. For instance, using a 500-μl/min flow rate and a 3-minute chromatographic gradient, Speedy-PASEF quantified 5,211 proteins from 2 μg of a mammalian cell-line standard at high quantitative accuracy and precision. We further used Speedy-PASEF to analyze blood plasma samples from a cohort of COVID-19 inpatients, using a 3-minute chromatographic gradient and alternating column regeneration on a dual pump system, for processing 398 samples per day. Speedy-PASEF delivered a comprehensive view of the COVID-19 plasma proteome, allowing classification of the patients according to disease severity and revealing plasma biomarker candidates. Speedy-PASEF thus facilitates acquisition of high-quality proteomes in large numbers.