Proteomics workflows have become increasingly successful in the characterization of complex proteomes in great depth. Application to large sample cohorts requires a high degree of reproducibility and data completeness, which makes DIA workflows interesting. DIA acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall, only a few percent of all incoming ions are isolated for mass analysis.
A collaboration between researchers from ETH Zürich, University of Belfast, University of Toronto and Bruker Daltonik were led by the Mann group at the Max Planck Institute for Biochemistry, Münich to develop the diaPASEF workflow for the timsTOF Pro. This scan mode samples up to 100% of the peptide precursor ion current in m/z and mobility windows by making use of the correlation of molecular weight and ion mobility in a trapped ion mobility device.
They optimized the diaPASEF schemes to balance selectivity, sensitivity and precursor coverage for fast gradients with the Evosep One. This typically require shorter mass spectrometry cycle times to achieve enough data points per peak for accurate quantification, thus a cycle time of 0.9 s were employed for the analysis of 60, 100 and 200 samples per day.
In triplicate analysis of 200 ng HeLa with the 60 samples per day method, they quantified on average 4,813 proteins per run and 5,183 in total with a median coefficient of variation of 5.8%. Remarkably, 4,255 proteins were quantified with a coefficient of variation of <20%. When they increased the throughput to 100 and 200 samples per day, more than 4,000 and 3,000 proteins in triplicate were still quantified, respectively.
The demonstrated diaPASEF workflow combines the advantages of DIA such as high degree of reproducibility and data completeness with the effective PASEF technology. Furthermore, they foresee that applications of diaPASEF could be extended to metabolites, lipids or other compound classes.
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