Use of Hybrid Data-Dependent and -Independent Acquisition Spectral Libraries Empowers Dual-Proteome Profiling

[Image: see text] In the context of bacterial infections, it is imperative that physiological responses can be studied in an integrated manner, meaning a simultaneous analysis of both the host and the pathogen responses. To improve the sensitivity of detection, data-independent acquisition (DIA)-based proteomics was found to outperform data-dependent acquisition (DDA) workflows in identifying and quantifying low-abundant proteins. Here, by making use of representative bacterial pathogen/host proteome samples, we report an optimized hybrid library generation workflow for DIA mass spectrometry relying on the use of data-dependent and in silico-predicted spectral libraries. When compared to searching DDA experiment-specific libraries only, the use of hybrid libraries significantly improved peptide detection to an extent suggesting that infection-relevant host-pathogen conditions could be profiled in sufficient depth without the need of a priori bacterial pathogen enrichment when studying the bacterial proteome. Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD017904 and PXD017945.