Evaluation of Data-Independent Acquisition-Based Phosphoproteomics Analysis in Mammalian and Bacterial Systems

Recent advancements in data-independent acquisition (DIA) workflows have greatly increased the depth and throughput of mass spectrometry-based proteomics. However, many of these advancements remain undercharacterized for phosphoproteomics, particularly in bacteria that have fewer protein phosphorylation events. We evaluated the impact of instrument/gradient length (Orbitrap Astral, 15 min; Orbitrap Exploris, 90 min), analysis tools (DIA-NN 1.9.2, DIA-NN 2.3, FragPipe, Spectronaut), and library search strategies (spectral-library versus library-free) on phosphoproteomic coverage and quantification in a human cell line and bacterial lysate. The 15 min Astral analysis identified similar numbers of phosphopeptides compared to a 90 min Exploris acquisition method across all analysis tools, demonstrating a substantial advantage in throughput. Within the Astral workflow, Spectronaut provided the highest phosphoproteome coverage, whereas DIA-NN 1.9.2, DIA-NN 2.3, and FragPipe exhibited less quantitative variation. We compared library-free and spectral-library search strategies using the Mycobacterium tuberculosis (Mtb) H37Rv strain. We observed greater phosphopeptide identifications yet different phosphopeptide profiles via library-free searches in comparison to spectral-library searches. When comparing Mtb harvested at different growth phases, phosphosite fold-changes were consistent, whereas statistical significance varied between tools. This work can be informative for workflow selection in DIA phosphoproteomics studies, especially for biological samples with low phosphorylation frequencies.