Considering Measurement Time and Depth in diaPASEF Plasma Proteomics.

Mass spectrometry (MS)-based plasma proteomics is a powerful approach to unraveling the biology or pathophysiology in large clinical cohorts. Implementation of data-independent acquisition in combination with ion mobility approaches (diaPASEF) has enabled high-throughput analysis with increasing proteomic depth. DIA-based methods are dependent upon experimental or in-silico-generated spectral libraries, yet there is a lack of consensus in the field about which library produces superior results in regard to protein and peptide identifications and quantifications. Here, we evaluated approaches for building a spectral library in plasma proteomics on a timsTOF HT system. Furthermore, the relationship between measurement time, library depth, and number of protein and peptide identification for high-throughput plasma proteomics applications was assessed. As expected, an increase in the measurement time invested in the spectral library enhanced the number of identifications. At the protein level, in silico libraries provided decreased depth compared to the most extensive experimental library. However, the experimental library enhanced the number of peptide identifications by 14% compared to that of the in silico library. With the field increasingly moving to peptide-centric approaches, an experimental library allows for deeper assessment in peptide-based studies.