Site-specific protein phosphorylation is a highly dynamic post-translational modification that influences protein activity and function.

With the advances in automated phosphopeptide enrichment and rapid MS analysis, the current bottleneck in phosphoproteomics workflows is the identification and quantification of phosphopeptides. With the generation of large phosphoproteomics raw files (>10Gb), using a spectral library-free approach for phosphopeptide identification present a significant challenge. In Spectronaut, search times can be reduced up to four-fold using a dedicated spectral library, however, the resulting depth of coverage using a spectral library is constrained by the library's quality and comprehensiveness.

Here, we used a wide panel of human cell lines, with and without stimulation, for single-shot phosphoproteomics analysis using an Evosep One LC system coupled to the Orbitrap Astral MS using narrow window data-independent acquisition (nDIA). Cell lysates were processed using PAC-based on-bead LysC/trypsin digestion followed by on-beads (Zr-IMAC HP) phosphopeptide enrichment in the KingFisher platform. From most cell lines including unstimulated HeLa, we quantified over 50,000 localized phosphosites (class I) with a median coefficient of variation below 20% with online LC gradients of 44 minutes (30SPD). The generated data was searched using Spectronaut for the generation of a comprehensive and high quality phosphopeptide library containing over 100,000 localized phosphosites (class I). This library was then benchmarked against a spectral library-free approach on a separate sample set. For biological insights, phosphorylation sites were quantified between the different cell line conditions to elucidate activated signaling pathways.

This project aims at addressing the challenge imposed by search time of phosphoproteomics data on the Orbitrap Astral MS by providing the largest experimental human phosphopeptide library. This resource reduces computational analysis time, enabling large-scale phosphoproteomics studies without compromising depth or requiring project-specific spectral libraries.