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.